Compositions including triciribine and bortezomib and derivatives thereof and methods of use thereof

ABSTRACT

This application relates to combination therapies including triciribine and related compounds and bortezomib and derivatives thereof analogs and compositions with reduced toxicity for the treatment and prevention of tumors, cancer, and other disorders associated with abnormal cell proliferation.

This application is a continuation-in-part of U.S. application Ser. No.11/096,082, filed Mar. 25, 2005, which claims the benefit of U.S.provisional patent application No. 60/557,599 filed Mar. 29, 2004, whichis incorporated herein by reference.

1. FIELD OF THE INVENTION

This application relates to combination therapies including triciribinecompounds and bortezomib and derivatives thereof and compositions withreduced toxicity for the treatment and prevention of tumors, cancer, andother disorders associated with abnormal cell proliferation.

2. BACKGROUND OF THE INVENTION

Cancer is an abnormal growth of cells. Cancer cells rapidly reproducedespite restriction of space, nutrients shared by other cells, orsignals sent from the body to stop reproduction. Cancer cells are oftenshaped differently from healthy cells, do not function properly, and canspread into many areas of the body. Abnormal growths of tissue, calledtumors, are clusters of cells that are capable of growing and dividinguncontrollably. Tumors can be benign (noncancerous) or malignant(cancerous). Benign tumors, tend to grow slowly and do not spread.Malignant tumors can grow rapidly, invade and destroy nearby normaltissues, and spread throughout the body.

Cancers are classified according to the kind of fluid or tissue fromwhich they originate, or according to the location in the body wherethey first developed. In addition, some cancers are of mixed types.Cancers can be grouped into five broad categories, carcinomas, sarcomas,lymphomas, leukemias, and myelomas, which indicate the tissue and bloodclassifications of the cancer. Carcinomas are cancers found in bodytissue known as epithelial tissue that covers or lines surfaces oforgans, glands, or body structures. For example, a cancer of the liningof the stomach is called a carcinoma. Many carcinomas affect organs orglands that are involved with secretion, such as breasts that producemilk. Carcinomas account for approximately eighty to ninety percent ofall cancer cases. Sarcomas are malignant tumors growing from connectivetissues, such as cartilage, fat, muscle, tendons, and bones. The mostcommon sarcoma, a tumor on the bone, usually occurs young adults.Examples of sarcoma include osteosarcoma (bone) and chondrosarcoma(cartilage). Lymphoma refers to a cancer that originates in the nodes orglands of the lymphatic system, whose job it is to produce white bloodcells and clean body fluids, or in organs such as the brain and breast.Lymphomas are classified into two categories: Hodgkin's lymphoma andnon-Hodgkin's lymphoma. Leukemia, also known as blood cancer, is acancer of the bone marrow that keeps the marrow from producing normalred and white blood cells and platelets. White blood cells are needed toresist infection. Red blood cells are needed to prevent anemia.Platelets keep the body from easily bruising and bleeding. Examples ofleukemia include acute myelogenous leukemia, chronic myelogenousleukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia.The terms myelogenous and lymphocytic indicate the type of cells thatare involved. Finally, myelomas grow in the plasma cells of bone marrow.In some cases, the myeloma cells collect in one bone and form a singletumor, called a plasmacytoma. However, in other cases, the myeloma cellscollect in many bones, forming many bone tumors. This is called multiplemyeloma.

Tumor induction and progression are often the result of accumulatedchanges in the tumor-cell genome. Such changes can include inactivationof cell growth inhibiting genes, or tumor suppressor genes, as well asactivation of cell growth promoting genes, or oncogenes. Hundreds ofactivated cellular oncogenes have been identified to date in animalmodels, however, only a small minority of these genes have proven to berelevant to human cancers (Weinberg et al., 1989, Oncogenes and theMolecular Origins of Cancer Cold Spring Harbor, N.Y.; Stanbridge andNowell, 1990, Cell 63: 867-874; Godwin et al., 1992, Oncogenes andantioncogenes in gynecological malignancies, in W J Hoskins, C A Perezand R C Young (eds), Gynecological oncology: principles and practice, pp87-116, Lippincott, Philadelphia). The activation of oncogenes in humancancers can result from factors such as increased gene copy number orstructural changes. These factors can cause numerous cellular effects,for example, they can result in overexpression of a gene product.Several oncogenes involved in human cancer can be activated through geneoverexpression.

It has become apparent that the successive genetic aberrations acquiredby cancer cells result in defects in regulatory signal transductioncircuits that govern normal cell proliferation, differentiation andprogrammed cell death (Hanahan et al., 2000, Cell 100: 57-700). This inturn results in fundamental defects in cell physiology which dictatemalignancy. These defects include: a) self sufficiency in growth signals(i.e. overexpression of growth factor receptor tyrosine kinases such asEGFR and aberrant activation of downstream signal transduction pathwayssuch as Ras/Raf/Mek/Erk ½ and Ras/PI3K/Akt), b) resistance toanti-growth signals (i.e. lower expression of TGFβ and its receptor), c)evading apoptosis (i.e. loss of proapoptotic p53; overexpression ofpro-survival Bcl-2; hyperactivation of survival pathways such as thosemediated by PI3K/Akt), d) sustained angiogenesis (i.e. high levels ofsecretion of VEGF) and f) tissue invasion and metastasis (i.e.extracellular proteases and prometastatic integrins) (Hanahan et al.,2000, Cell 100: 57-700).

Receptor tyrosine kinases such as EGFR, ErbB2, VEGFR and insulin-likegrowth factor I receptor (IGF-1R) are intimately involved in thedevelopment of many human cancers including colorectal pancreatic,breast and ovarian cancers (Khaleghpour et al., 2004, Carcinogenesis 25:241-8; Sekharam et al., 2003, Cancer Res 63: 7708-16). Binding ofligands such as EGF, VEGF and IGF-1 to their receptors promotesstimulation of the intrinsic tyrosine kinase activity,autophosphorylation of specific tyrosines in the cytoplasmic domain ofthe receptors and recruitment of signaling proteins that trigger avariety of complex signal transduction pathways (Olayioye et al., 2000,Embo J 19: 3159-3167, Porter et al., 1998, Oncogene 17: 1343-52). Thisin turn leads to the activation of many tumor survival and oncogenicpathways such as the Ras/Raf/Mek/Erk ½, JAK/STAT3 and PI3K/Akt pathways.Although all three pathways have been implicated in colon, pancreatic,breast and ovarian oncogenesis, those that are mediated by Akt have beenshown to be critical in many steps of malignant transformation includingcell proliferation, anti-apoptosis/survival, invasion and metastasis andangiogenesis (Datta et al., 1999, Genes Dev 13: 2905-2927).

Akt is a serine/threonine protein kinase (also known as PKB), which has3 family members Akt1, Akt2 and Akt3. Stimulation of cells with growthor survival factors results in recruitment to the receptors of the lipidkinase phosphoinositide-3-OH-kinase (PI3K) which phosphorylatesphosphoinositol-4,5-biphosphate (PIP₂) to PIP₃ which recruits Akt to theplasma membrane where it can be activated by phosphorylation on Thr308and Ser473 (Akt1), Thr308 and Ser474 (Akt2) and Thr308 and Ser472 (Akt3)(Datta et al., 1999, Genes Dev 13: 2905-2927). Thus, PI3K activates Aktby phosphorylating PIP2 and converting to PIP3. The phosphatase PTENdephosphorylates PIP3 to PIP2 and hence prevents the activation of Akt.

The majority of human cancers contain hyperactivated Akt (Datta et al.,1999, Genes Dev 13: 2905-2927; Bellacosa et al., 1995. Int J Cancer 64:280-285; Sun et al., 2001, Am J Pathol 159: 431-437). In particular, Aktis overexpressed and/or hyperactivated in 57%, 32%, 27% and 36% of humancolorectal, pancreatic, breast and ovarian cancers, respectivel (Roy etal., 2002, Carcinogenesis 23: 201-205; Altomare et al., 2003, J CellBiochem 88: 470-476; Sun et al., 2001, Cancer Res 61: 5985-5991; Stal etal., 2003, Breast Cancer Res 5: R37-R44; Cheng et al., 1992, Proc NatlAcad Sci U S A 89: 9267-9271; Yuan et al., 2000, Oncogene 19:2324-2330). Hyperactivation of Akt is due to amplification and/oroverexpression of Akt itself as well as genetic alterations upstream ofAkt including overexpression of receptor tyrosine kinases and/or theirligands (Khaleghpour et al., 2004, Carcinogenesis 25: 241-248; Sekharamet al., 2003, Cancer Res 63: 7708-7716; Cohen et al., 1998, Biochem SocSymp 63: 199-210; Muller et al., 1998, Biochem Soc Symp 63: 149-157;Miller et al., 1995, J Virol 69: 4390-4398; Slamon et al., 1987, Science235: 177-182; Andrulis et al., 1998, J Clin Oncol 16: 1340-1349) anddeletion of the phosphatase PTEN. Proof-of-concept of the involvement ofAkt in oncogenesis has been demonstrated preclinically by showing thatectopic expression of Akt induces malignant transformation and promotescell survival (Sun et al., 2001, Am J Pathol 159: 431-437; Cheng et al.,1997, Oncogene 14: 2793-2801) and that disruption of Akt pathwaysinhibits cell growth and induces apoptosis (Jetzt et al., 2003, CancerRes 63: 6697-6706).

Current treatments of cancer and related diseases have limitedeffectiveness and numerous serious unintended side effects. Despitedemonstrated clinical efficacy of many anti-cancer drugs, severesystemic toxicity often halts the clinical development of promisingchemotherapeutic agents. Further, overexpression of receptor tyrosinekinases such as EGFR and their ligands such as IGF-1, Akt overexpressionand/or loss of PTEN (all of which result in hyperactivation of Akt) areassociated with poor prognosis, resistance to chemotherapy and shortenedsurvival lime of cancer patients. Current research strategies emphasizethe search for effective therapeutic modes with less risk.

Thus, a combination of a triciribine compound or a derivative thereofand bortezomib or a salt or derivative thereof holds promise as apotential combination therapy for treating tumors, cancer, and abnormalcell proliferation.

3. SUMMARY OF THE INVENTION

The present invention provides novel therapeutic regimens oftriciribine, triciribine phosphate and related compounds in combinationwith bortezomib and derivatives thereof and derivatives thereof to treattumors or cancer in a subject while limiting systemic toxicity. Theinvention is based on the discovery that tumors or cancers, whichoverexpress Akt kinase are particularly sensitive to the cytotoxiceffects of TCN and related compounds and a synergistic affect wouldarise with a combination of bortezomib salts or derivatives thereof andderivatives thereof. The inventors have determined, contrary to theprior art and experience, how to successfully use triciribine andbortezomib and derivatives thereof and derivatives thereof to treattumors and cancer by one or a combination of (i) administeringtriciribine and bortezomib and salts or derivatives thereof andderivatives thereof to patients who exhibit enhanced sensitivity to thedrug; (ii) use of a described dosage level that minimizes the toxicityof the drugs but yet still exhibits efficacy; or (iii) use of adescribed dosage regimen that minimizes the toxicity of the drugs.

In one illustrative embodiment of the invention, the inventionencompasses a composition including:

(i) a compound of the formula I-IV:

wherein each R₂′, R₃′ and R₅′ are independently hydrogen; optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R₂′, R₃′ or R₅′ is independently H or mono-,di-, or tri-phosphate;

wherein R^(x) and R^(y) are independently hydrogen, optionallysubstituted phosphate; acyl (including lower acyl); amide,alkyl(including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl;

(ii) a compound of formula V:

wherein

R₁, R₂, R₃, R₄, and R₅ each are independently H, optionally halogenated,substituted straight chained, branched or cyclic alkyl (including loweralkyl), alkoxyl, alkenyl, or alkynyl, aryl, CO-alkyl, CO-alkenyl,CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl,CO-substituted aryl, sulfonyl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl;

and

(iii) a pharmaceutically acceptable carrier.

In another illustrative embodiment of the invention, the inventionencompasses a composition including TCN, TCN-P, TCN-PM or a combinationthereof and bortezomib or a salt or derivative thereof.

In another illustrative embodiment, the invention encompasses a methodof treating a tumor or cancer in a mammal including administering to themammal an effective amount of a composition including:

(i) a compound of formula I-IV:

-   -   wherein each R₂′, R₃′ and R₅′ are independently hydrogen,        optionally substituted phosphate or phosphonate (including        mono-, di-, or triphosphate or a stabilized phosphate prodrug);        acyl (including lower acyl); alkyl (including lower alkyl);        amide, sulfonate ester including alkyl or arylalkyl; sulfonyl,        including methanesulfonyl and benzyl, wherein the phenyl group        is optionally substituted with one or more substituents as for        example as described in the definition of an aryl given herein;        optionally substituted arylsulfonyl; a lipid, including a        phospholipid; an amino acid; a carbohydrate; a peptide; or        cholesterol; or other pharmaceutically acceptable leaving group        that, in vivo, provides a compound wherein R₂′, R₃′ and R₅′ is        independently H or mono-, di- or tri-phosphate;    -   wherein R^(x) and R^(y) are independently hydrogen, optionally        substituted phosphate; acyl (including lower acyl); amide, alkyl        (including lower alkyl); aromatic, polyoxyalkylene such as        polyethyleneglycol, optionally substituted arylsulfonyl; a        lipid, including a phospholipid; an amino acid; a carbohydrate;        a peptide; or cholesterol; or other pharmaceutically acceptable        leaving group. In one embodiment, the compound is administered        as a 5′-phosphoether lipid or a 5′-ether lipid; and    -   R₁ and R₂ each are independently H, optionally substituted        straight chained, branched or cyclic alkyl (including lower        alkyl), alkenyl, or alkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl,        CO-aryl or heteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl,        CO-substituted aryl, sulfonyl, alkylsulfonyl, arylsulfonyl,        aralkylsulfonyl; and

(ii) a compound of formula V:

wherein

R₁, R₂, R₃, R₄, and R₅ each are independently H, optionally halogenated,Substituted straight chained, branched or cyclic alkyl (including loweralkyl), alkoxyl, alkenyl, or alkynyl, aryl, CO-alkyl, CO-alkenyl,CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl,CO-substituted aryl, sulfonyl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl.

In another illustrative embodiment, the invention encompasses a methodof treating a tumor or cancer in a mammal including administering to themammal an effective amount of a composition including TCN, TCN-P, TCN-PMor a combination thereof and bortezomib or a salt or derivative thereof.

Methods are useful to treat tumors and cancers that are particularysusceptible to the toxic effects of TCN, TCN-P, TCN-PM and/or relatedcompounds. In another embodiment, methods are provided for treating atumor in a mammal, particularly a human that includes (i) obtaining abiological sample from the tumor; (ii) determining whether the tumoroverexpresses an Akt kinase, and (iii) treating the tumor thatoverexpresses Akt kinase with triciribine, triciribine phosphate or arelated compound in combination with bortezomib or a salt or derivativethereof. In another embodiment, the level of Akt kinase expression canbe determined by assaying the tumor or cancer for the presence of aphosphorylated Akt kinase, for example, by using an antibody that candetect the phosphorylated form. In another embodiment, the level of Aktexpression can be determined by assaying a tumor or cancer cellsobtained from a subject and comparing the levels to a control tissue. Incertain embodiments, the Akt can be overexpressed at least 2, 2.5, 3 or5 fold in the cancer sample compared to the control. In certainembodiments, the overexpressed Akt kinase can be a hyperactivated andphosphorylated Akt kinase.

In another aspect of the present invention, dosing regimens are providedthat limit the toxic side effects of TON and related compounds. Inanother embodiment, such dosing regimens minimize or eliminate toxicside effects, including, but not limited to, hepatoxicity,thrombocytopenia, hyperglycemia, vomiting, hypocalcemia, anemia,hypoalbunemia, myelosuppression, hypertriglyceridemia, hyperamylasemia,diarrhea, stomachitis and/or fever. In another embodiment, theadministration of TCN, TCN-P, TCN-PM or related compounds provides atleast a partial, such as at least 15, 20 or 30%, or complete response invivo in at least 15, 20, or 25% of the subjects.

In another embodiment, a method is provided to treat a subject which hasbeen diagnosed with a tumor by administering to the subject an effectiveamount of TCN, TCN-P, TCN-PM or a related compound and bortezomib andderivatives thereof analogs according to a dosing schedule that includesadministering the drug approximately one time per week for approximatelythree weeks followed by a one week period wherein the drug is notadministered. In another embodiment, methods are provided to treat tumoror cancer in a subject by administering to the subject a dosing regimenof 10 mg/m² or less of TCN, TCN-P, TCN-PM or a related compound andbortezomib and derivatives thereof analogs each one time per week. Inanother embodiment, the triciribine compound and bortezomib andderivatives thereof analogs can be administered as a single bolus doseover a short period of time, for example, about 5, 10 or 15 minutes. Infurther embodiments, dosing schedules are provided in which thetriciribine compound and bortezomib and derivatives thereof analogs areadministered via continuous infusion for at least 24, 48, 72, 96, or 120hours. In certain embodiments, the continuous administration can berepeated at least once a week, once every two weeks and/or once a month.In other embodiments, the triciribine compound and bortezomib andderivatives thereof analogs can be administered at least once everythree weeks. In further embodiments, the compounds can be administeredat least once a day for at least 2, 3, 4 or 5 days.

In further embodiments, the triciribine compound and bortezomib andderivatives thereof analogs as disclosed herein can be administered topatients in an amount that is effective in causing tumor regression. Theadministration the triciribine compound and bortezomib and derivativesthereof analogs can provide at least a partial, such as at least 15, 20or 30%, or complete response in vivo in at least 15-20% of the subjects.In certain embodiments, at least 2, 5, 10, 15, 20, 30 or 50 mg/m² of thetriciribine compound and bortezomib and derivatives thereof analogsdisclosed herein can be administered to a subject. The administration ofthe triciribine compound and bortezomib and derivatives thereof analogscan be conducted according to any of the therapeutic regimens disclosedherein. In particular embodiments, the dosing regimen can includeadministering less than 20 mg/m² of the triciribine compound andbortezomib and derivatives thereof analgos. In one embodiment, less than10 mg/m² of the triciribine compound and bortezomib and derivativesthereof analogs can be administered once a week. In further embodiments,dosages of or less than 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² ofthe triciribine compound and bortezomib and derivatives thereof analogscan be administered to a subject. In another embodiment, less than 10mg/m² can be administered to a subject via continuous infusion for atleast five days. In particular embodiments, the triciribine compound andbortezomib and derivatives thereof analogs as disclosed herein can beused for the treatment of pancreatic, prostate, colo-rectal and/orovarian cancer.

In another embodiment, the triciribine compound and bortezomib andderivatives thereof analogs and/or therapeutic regimens of the presentinvention can be used to prevent and/or treat a carcinoma, sarcoma,lymphoma, leukemia, and/or myeloma. In other embodiments of theinvention, the triciribine compound and bortezomib and derivativesthereof analogs can be used to treat solid tumors. In still furtherembodiments, the triciribine compound and bortezomib and derivativesthereof analogs and compositions disclosed herein can be used for thetreatment of a tumor or cancer, such as, but not limited to cancer ofthe following organs or tissues: breast, prostate, bone, lung, colon,including, but not limited to colorectal, urinary, bladder, non-Hodgkinlymphoma, melanoma, kidney, renal, pancreas, pharnx, thyroid, stomach,brain, and/or ovaries. In a particular embodiment, the triciribinecompound and bortezomib and derivatives thereof analogs can be used forthe treatment of pancreatic, breast, colorectal and/or ovarian cancer.In further embodiments of the present invention, the triciribinecompound and bortezomib and derivatives thereof analogs disclosed hereincan be used in the treatment of angiogenesis-related diseases. Incertain embodiments, methods are provided to treat leukemia viacontinuous infusion of the triciribine compound and bortezomib andderivatives thereof analogs via continuous infusion for at least 24, 48,72 or 96 hours. In other embodiments, the continuous infusion can berepeated, for example, at least once every two, three or four weeks.

In a particular embodiment, there is provided a method for the treatmentof tumors, cancer, and others disorders associated with an abnormal cellproliferation in a host, the method comprising administering to the hostan effect amount of the triciribine compound and bortezomib andderivatives thereof analogs optionally in combination with apharmaceutically acceptable carrier.

In one aspect, the triciribine compound and bortezomib and derivativesthereof analogs and compositions can be administered in combination andcan form part of the same composition, or be provided as a separatecomposition for administration at the same time or a different time.

In other embodiments, the triciribine compound and bortezomib andderivatives thereof analogs as disclosed herein can be used to treattumors or cancers resistant to drugs, including the embodiments oftumors or cancers and drugs disclosed herein. In one embodiment, thetriciribine compound and bortezomib and derivatives thereof analogs asdisclosed herein is administered in an effective amount for thetreatment of a patient with a drug resistant tumor or cancer, forexample, multidrug resistant tumors or cancer including, but not limitedto, those resistant to taxol alone, rapamycin, tamoxifen, cisplatin,and/or gefitinib (iressa).

In certain embodiments, a method is provided including administering toa host in need thereof an effective amount of a triciribine compound andbortezomib and derivatives thereof analogs disclosed herein, orpharmaceutical composition comprising a triciribine compound andbortezomib and derivatives thereof analogs, in an effective amount forthe treatment of the treatment of tumors, cancer, and others disordersassociated with an abnormal cell proliferation in a host.

In another embodiment, a method for the treatment of a tumor or canceris provided including an effective amount of a compound disclosedherein, or a salt, isomer, prodrug or ester thereof, to an individual inneed thereof, wherein the cancer is for example, carcinoma, sarcoma,lymphoma, leukemia, or myeloma. The compound, or salt, isomer, prodrugor ester thereof, is optionally provided in a pharmaceuticallyacceptable composition including the appropriate carriers, such aswater, which is formulated for the desired route of administration to anindividual in need thereof. Optionally the compound is administered incombination or alternation with at least one additional therapeuticagent for the treatment of tumors or cancer.

Also within the scope of the invention is the use of a compounddisclosed herein or a salt, prodrug or ester thereof in the treatment ofa tumor or cancer, optionally in a pharmaceutically acceptable carrier;and the use of a triciribine compound and bortezomib and derivativesthereof analogs disclosed herein or a salt, prodrug or ester thereof inthe manufacture of a medicament for the treatment of cancer or tumor,optionally in a pharmaceutically acceptable carrier.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the identification of API-2 (triciribine) as acandidate of Akt inhibitor from the NCI Diversity Set. A illustrates thechemical structure of API-2 (triciribine). B demonstrates that API-2inhibits phosphorylation levels of AKT2 in AKT2-transformed NIH3T3cells. Wile type AKT2-transformed NIH3T3 cells were treated with API-2(1 μM) for indicated times and subjected to immunoblotting analysis withanti-phospho-Akt-T308 and -S473 antibodies (top and middle panels). Thebottom panel shows expression of total AKT2. In C, it is shown thatAPI-2 inhibits three isoforms of Akt. HEK293 cells were transfected withHA-Akt1, -AKT2 and -AKT3 and treated with API-2 (1 μM) or wortmannin (15μM) prior to EGF stimulation, the cells were lysed andimmunoprecipitated with anti-HA antibody. The immunoprecipitates weresubjected to in vitro kinase assay (top) and immunoblotting analysiswith anti-phospho-Akt-T308 (bottom) antibody. Middle panel showsexpression of transfected Akt1, AKT2 and AKT3. D illustrates that API-2did not inhibit Akt in vitro. In vitro kinase assay of constitutivelyactive AKT2 recombinant protein in a kinase buffer containing 1 μM API-2(lane 3).

FIG. 2 demonstrates that API-2 does not inhibit PI3K, PDK1 and theclosely related members of AGC kinase family. A demonstrates an in vitroPI3K kinase assay. HEK293 cells were serum-starved and treated withAPI-2 (1 μM) or Wortmannin (15 μM) fro 30 minutes prior to EGFstimulation. Cells were lysed and immunoprecipitated with anti-p110αantibody. The immunoprecipitates were subjected to in vitro kinase assayusing PI-4-P as substrate. B illustrates the effect of API-2 on in vitroPDK1 activation (top panel), closed circles show inhibition by API-2.Open circles show inhibition by the positive control staurosporine,which is a potent PDK1 inhibitor (IC50=5 nM). Bottom panels areimmunoblotting analysis of HEK293 cells that were transfected withMyc-PDK1 and treated with wortmannin or API-2 prior to EGF stimulation.The immunoblots were detected with indicated antibodies. C illustratesan immunoblot analysis of phosphorylation levels of PKCα withanti-phospho-PKCα-T638 (top) and total PKCα (bottom) antibodiesfollowing treatment with API-2or a nonselective PKC inhibitor Ro31-8220.D shows an in vitro SGK kinase assay. HEK293 cells were transfected withHA-SGK and treated with API-2 or wortmannin prior to EGF stimulation. Invitro kinase was performed with HA-SGK immunoprecipitates using MBP assubstrate (top). Bottom panel shows the expression of transfectedHA-SGK. E illustrates the results of a PKA kinase assay. Immuno-purifiedPKA was incubated in ADB buffer (Upstate Biotechnology Inc) containingindicated inhibitors (API-2 or PKAI) and substrate Kemptide. The kinaseactivity was quantified. In F, a western blot is shown. OVCAR3 cellswere treated with API-2 for indicated times. Cell lysates wereimmunoblotted with indicated anti-phospho-antibodies (panels 1-4) andanti-actin antibody (bottom).

FIG. 3 demonstrates that API-2 inhibits Akt activity and cell growth andinduces apoptosis in human cancer cells with elevated Akt. A is awestern blot, following treatment with API-2, phosphorylation levels ofAkt were detected with anti-phospho-Akt-T308 antibody in indicated humancancer cell lines. The blots were reprobed with anti-total Akt antibody(bottom panels). In B, a cell proliferation assay is shown. Cell linesas indicated in the figure were treated with different doses of API-2for 24 h and 48 h and then analyzed with CellTiter 96 Cell ProliferationAssay kit (Promega). C provides an apoptosis analysis. Cells weretreated with API-2 and stained with annexin V and PI and analyzed byFACScan.

FIG. 4 shows that API-2 inhibits downstream targets of Akt and exhibitsanti-tumor activity in cancer cell lines with elevated Akt in mousexenograft. In A, it is demonstrated that API-2 inhibits Aktphosphorylation of tuberin, Bad, AFX and GSK-3β. Following treatmentwith API-2, OVAR3 cells were lysed and immunoblotted with indicatedantibodies. B shows that API-2 inhibits tumor growth. Tumor cells weresubcutaneously injected into nude mice with low level of Akt cells onleft side and elevated level of Akt cells on right side. When the tumorsreached an average size of about 100-150 mm³, animals were treated witheither vehicle or 1 mg/kg/day API-2. Each measurement represents anaverage of 10 tumors. C illustrates a representation of the mice withOVCAR3 (right) and OVCAR5 (left) xenograft treated with API-2 or vehicle(control). D shows examples of tumor size (bottom) and weight (top) atthe end of experiment. In E, immunoblot analysis of tumor lysates wasperformed with anti-phospho-Akt-S473 (top) and anti-AKT2 (bottom)antibodies in OVCAR-3-derived tumors that were treated (T3 and T4) anduntreated (T1 and T2) with API-2.

FIG. 5 shows that API-2 (triciribine) inhibits Akt kinase activity invitro. In vitro kinase assay was performed with recombinant of PDK1 andAkt in a kinase buffer containing phosphatidylinositol-3,4,5-P3 (PIP3),API-2 and histone H2B as substrate. After incubation of 30 min, thereactions were separated by SDS-PAGE and exposed in a film.

FIG. 6 provides the mRNA and amino acid sequence of human Akt1,restriction enzyme sites are also noted.

FIG. 7 provides the mRNA and amino acid sequence of human Akt2restriction enzyme sites are also noted.

FIG. 8 provides the mRNA and amino acid sequence of human Akt3restriction enzyme sites are also noted.

FIG. 9 shows bortezomib synergistically enhances the effect of API-2(TCN) on the survival myeloma cells. H929 cells (multiple myelomaderived) were treated with 0, 2.5, 5, 10, 20, or 40 nM bortezomib aloneor in the presence of 10 μM API-2, or with 0, 2.5, 5, 10, 20, or 40 μMAPI-2 alone. Cell survival rates were calibrated and graphed (FIG. 9A).U266 cells (multiple myeloma derived) were treated with 0, 2.5, 5, 10,20, or 40 μM API-2 alone or in the presence of 10 nM API-2, or with 0,2.5, 5, 10, 20, or 40 μM bortezomib alone. Cell survival rates werecalibrated and graphed (FIG. 9B).

FIG. 10 shows the synergistic effect of bortezomib and API-2 (TCN) onmantle cell lymphoma cells. Jeko-1 cells (mantel cell lymphoma derived)were treated with 0, 2.5, 5, 10, 20, 40, or 80 nM bortezomib, alone orin the presence of 5 μM API-2, or with 0, 1.25, 2.5, 5, 10, 20, or 40 μMAPI-2. Cell survival rates were calibrated and graphed.

5. DETAILED DESCRIPTION OF THE INVENTION

The inventors have determined, contrary to the prior art and experience,how to successfully use triciribine compounds in combination withbortezomib and derivatives thereof analogs to treat tumors and cancer byone or a combination of (i) administering triciribine and bortezomib andderivatives thereof analogs only to patients which according to adiagnostic test described below, exhibit enhanced sensitivity to thetriciribine compound and/or the bortezomib and derivatives thereof; (ii)using a described dosage level that minimizes the toxicity of thetriciribine compound and/or the bortezomib and derivatives thereofanalogs but yet still exhibits efficacy; or (iii) using a describeddosage regimen that minimizes the toxicity of the triciribine compoundand/or the bortezomib and derivatives thereof analogs.

5.1. Definitions

As used herein, the term “compounds of the invention” refers tocompounds of formula I-V and combinations thereof.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth, i.e., proliferative disorders. Examples ofsuch proliferative disorders include cancers such as carcinoma,lymphoma, blastoma, sarcoma, and leukemia, as well as other cancersdisclosed herein. More particular examples of such cancers includebreast cancer, prostate cancer, colon cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, cervical cancer, ovarian cancer, livercancer, e.g., hepatic carcinoma, bladder cancer, colorectal cancer,endometrial carcinoma, kidney cancer, and thyroid cancer.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CMS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); pancreatic cancer; retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; sarcoma; skin cancer;stomach cancer; testicular cancer; uterine cancer; cancer of the urinarysystem, as well as other carcinomas and sarcomas.

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor. The solid tumor mass, if present,may be a primary tumor mass. A primary tumor mass refers to a growth ofcancer cells in a tissue resulting from the transformation of a normalcell of that tissue. In most cases, the primary tumor mass is identifiedby the presence of a cyst, which can be found through visual orpalpation methods, or by irregularity in shape, texture or weight of thetissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography), or by needle aspirations. The use of these lattertechniques is more common in early detection. Molecular and phenotypicanalysis of cancer cells within a tissue will usually confirm if thecancer is endogenous to the tissue or if die lesion is due to metastasisfrom another site.

The term alkyl, as used herein, unless otherwise specified, includes asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of for example C₁ to C₂₄, and specifically includes methyl,trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The alkyl is optionally substituted, e.g., withsubstituents such as halo (F, Cl, Br or I), (e.g. CF₃, 2-Br-ethyl, CH₂F,CH₂Cl, CH₂CF₃ or CF₂CF₃), hydroxyl (e.g. CH₂OH), amino (e.g. CH₂NH₂,CH₂NHCH₃ or CH₂N(CH₃)₂), alkylamino, arylamino, alkoxy, aryloxy, nitro,azido (e.g. CH₂N₃), cyano (e.g. CH₃CN), sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term lower alkyl, as used herein, and unless otherwise specified,refers to a C₁ to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl) alkyl group, including bothsubstituted and unsubstituted forms.

The term alkylamino or arylamino includes an amino group that has one ortwo alkyl or aryl substituents, respectively.

The term amino acid includes naturally occurring and synthetic α, β, γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutamyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl,β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl. When the term amino acid is used, it is considered to be aspecific and independent disclosure of each of the esters of a naturalor synthetic amino acid, including but not limited to α, β, γ or δglycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginineand histidine in the D and L-configurations.

The term “protected” as used herein and unless otherwise definedincludes a group that is added to an oxygen, nitrogen, sulfur orphosphorus atom to prevent its further reaction or for other purposes. Awide variety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis (see Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, Inc., New York,N.Y., 1999).

The term aryl, as used herein, and unless otherwise specified, includesphenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl group isoptionally substituted with moieties such as halo, hydroxyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, 3^(rd) Ed., 1999.

The term alkaryl or alkylaryl includes an alkyl group with an arylsubstituent. The term aralkyl or arylalkyl includes an aryl group withan alkyl substituent.

The term halo, as used herein, includes chloro, bromo, iodo, and fluoro.

The term acyl includes a carboxylic acid ester in which the non-carbonylmoiety of the ester group is selected from straight, branched, or cyclicalkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkylincluding benzyl, aryloxyalkyl such as phenoxymethyl, aryl includingphenyl optionally substituted with halogen, C₁ to C₄ alkyl or C₁ to C₄alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the estersoptimally comprise a phenyl group. The term “lower acyl” refers to anacyl group in which the non-carbonyl moiety is lower alkyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” with respect to enantiomeric purity, refers to acomposition that includes at least 85% or 90% by weight, preferably 95%to 98% by weight, and even more preferably 99% to 100% by weight, of thedesignated enantiomer. In a preferred embodiment, in the methods andcompounds of this invention, the compounds are substantially free ofother enantiomers.

Similarly, the term “isolated” refers to a compound composition thatincludes at least 85% or 90% by weight; preferably 95% to 98% by weight,and even more preferably 99% to 100% by weight, of the compound, theremainder comprising other chemical species or enantiomers.

The term “independently” is used herein to indicate that the variable,which is independently applied, varies independently from application toapplication. Thus, in a compound such as R″XYR″, wherein R″ is“independently carbon or nitrogen,” both R″ can be carbon, both R″ canbe nitrogen, or one R″ can be carbon and the other R″ nitrogen.

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a compound, which, upon administration to a patient, providesthe compound. Pharmaceutically acceptable salts include those derivedfrom pharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of die present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, animated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated to produce theactive compound.

The term “pharmaceutically acceptable esters” as used herein, unlessotherwise specified, includes those esters of compounds, which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of hosts without undue toxicity, irritation, allergicresponse and the like, are commensurate with a reasonable benefit/riskratio, and are effective for their intended use.

The term “subject” as used herein refers to an animal, preferably amammal, most preferably a human. Mammals can include non-human mammals,including, but not limited to, pigs, sheep, goats, cows (bovine), deer,mules, horses, monkeys and other non-human primates, dogs, cats, rats,mice, rabbits or any other known or disclosed herein.

5.2. Compounds of the Invention

The present invention provides for the use of TCN, TCN-P, TCN-PM andrelated compounds in combination with bortezomib and derivatives thereofanalogs for use in particular therapeutic regimens for the treatment ofproliferative disorders.

As used herein and unless otherwise indicated, the term “triciribinecompounds” and “triciribine and related compounds” refers to compoundshaving the following structures:

wherein each R2′, R3′ and R5′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with substituents asfor example as described in the definition of an aryl given herein;optionally substituted arylsulfonyl: a lipid, including a phospholipid;an amino acid; a carbohydrate; a peptide; or cholesterol; or otherpharmaceutically acceptable leaving group that, in vivo, provides acompound wherein R2′, R3′ or R5′ is independently H or mono-, di- ortri-phosphate; wherein R^(x) and R^(y) are independently hydrogen,optionally substituted phosphate; acyl (including lower acyl); amide,alkyl (including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In one embodiment, R2′ and R3′ are hydrogen. In another embodiment, R2′and R5′ are hydrogen. In yet another embodiment, R2′, R3′ and R5′ arehydrogen. In yet another embodiment, R2′, R3′, R5′, R1 and R2 arehydrogen.

In another embodiment, the triciribine compound has the followingstructure:

-   -   wherein R₃ is H, optionally substituted straight chained,        branched or cyclic alkyl (including lower alkyl), alkenyl, or        alkynyl, NH₂, NHR⁴, N(R⁴)₂, aryl, alkoxyalkyl, aryloxyalkyl, or        substituted aryl; and    -   Each R⁴ independently is H, acyl including lower acyl, alkyl        including lower alkyl such as but not limited to methyl, ethyl,        propyl and cyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy,        alkoxyalkyl, hydroxyalkyl, or aryl. In a subembodiment, R₃ is a        straight chained C1-11 alkyl, iso-propyl, t-butyl, or phenyl.

In one embodiment, the triciribine compounds provided herein have thefollowing structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

-   -   wherein R₆ is H, alkyl, (including lower alkyl) alkenyl,        alkynyl, alkoxyalkyl, hydroxyalkyl, arylalkyl, cycloalkyl, NH₂,        NHR⁴, NR⁴R⁴, CF₃, CH₂OH, CH₂F, CH₂Cl, CH₂CF₃, C(Y³)₃,        C(Y³)₂C(Y³)₃), C(═O)OH, C(═O)OR⁴, C(═O)-alkyl, C(═O)-aryl,        C(═O)-alkoxyalkyl, C(═O)NH₂, C(═O)NHR⁴, C(═O)N(R⁴)₂, where each        Y³ is independently H or halo; and    -   each R⁴ independently is H, acyl including lower acyl, alkyl        including lower alkyl such as but not limited to methyl, ethyl,        propyl and cyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy,        alkoxyalkyl, hydroxyalkyl, or aryl.

In a subembodiment, R₆ is ethyl, CH₂CH₂OH, or CH₂-phenyl.

In another embodiment, the triciribine compounds provided herein havethe following structure:

-   -   wherein R₇ is H, halo, alkyl (including lower alkyl), alkenyl,        alkynyl, alkoxy, alkoxyalkyl, hydroxyalkyl, cycloalkyl, nitro,        cyano, OH, OR⁴, NH₂, NHR⁴, NR⁴R⁴, SH, SR⁴, CF₃, CH₂OH, CH₂F,        CH₂Cl, CH₂CF₃, C(Y³)₃, C(Y³)₂C(Y³)₃, C(═O)OH, C(═O)OR⁴,        C(═O)-alkyl, C(═O)-aryl, C(═O)-alkoxyalkyl, C(═O)NH₂, C(═O)NHR⁴,        C(═O)N(R⁴)₂, or N₃, where each Y³ is independently H or halo;        and    -   each R¹ independently is H, acyl including lower acyl, alkyl        including lower alkyl such as but not limited to methyl, ethyl,        propyl and cyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy,        alkoxyalkyl, hydroxyalkyl.

In a subembodiment, R₇ is methyl, ethyl, phenyl, chloro or NH₂.

In another embodiment, the triciribine compounds provided herein havethe following structure:

In another embodiment, the triciribine compounds provided herein havethe following/structure:

As used herein and unless otherwise indicated, the term “bortezomib andderivatives thereof” refers to a compound of formula V:

wherein

R₁, R₂, R₃, R₄ and R₅ each are independently H, optionally halogenated,substituted straight chained, branched or cyclic alkyl (including loweralkyl), alkoxyl, alkenyl, or alkynyl, aryl, CO-alkyl, CO-alkenyl,CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl,CO-substituted aryl, sulfonyl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl.

In one embodiment, the bortezomib and derivatives thereof have thefollowing structure of formula VI:

whereinR₁, R₂, R₃, R₄, and R₅ each are independently H, optionally halogenated,substituted straight chained, branched or cyclic alkyl (including loweralkyl), alkoxyl, alkenyl, or alkynyl, aryl, CO-alkyl, CO-alkenyl,CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl,CO-substituted aryl, sulfonyl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl.

In another embodiment, the bortezomib and derivatives thereof analogshave the following structure of formula VI:

It is to be understood that the compounds disclosed herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomer ordiastereomeric mixtures. It is understood that the disclosure of acompound herein encompasses any racemic, optically active, polymorphic,or steroisomeric form, or mixtures thereof, which preferably possessesthe useful properties described herein, it being well known in the arthow to prepare optically active forms and how to determine activityusing the standard tests described herein, or using other similar testswhich are will known in the art. Examples of methods that can be used toobtain optical isomers of the compounds include the following:

-   -   i) physical separation of crystals—a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization—a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions—a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme    -   iv) enzymatic asymmetric synthesis—a synthetic technique whereby        at least one step of the synthesis uses an enzymatic reaction to        obtain an enantiomerically pure or enriched synthetic precursor        of the desired enantiomer;    -   v) chemical asymmetric synthesis—a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce assymetry (i.e., chirality) in the        product, which may be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations—a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations—a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions—this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors—a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography—a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase. The stationary phase can be made of chiral material or        the mobile phase can contain an additional chiral material to        provoke the differing interactions;    -   xi) chiral gas chromatography—a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents—a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvents;    -   xiii) transport across chiral membranes—a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane which allows only one        enantiomer of the racemate to pass through.

In some embodiments, triciribine, triciribine phosphate (TCN-P),triciribine 5′-phosphate (TCN-P), triciribine 5′-phosphate monohydrate(TCN-PM) or the DMF adduct of triciribine (TCN-DMF) are provided. TCNcan be synthesized by any technique known to one skilled in the art, forexample, as described in Tetrahedron Letters, 49: 4757-4760 (1971).TCN-P can be prepared by any technique known to one skilled in the art,for example, as described in U.S. Pat. No. 4,123,524. The synthesis ofTCN-DMF is described, for example, in INSERM, 81: 37-82 (1978). Othercompounds related to TCN as described herein can be synthesized, forexample, according to the methods disclosed in Gudmundsson et al., 2001,Nucleosides Nucleotides Nucleic Acids 20: 1823-1830; Porcari et al.,2000, J Med Chem 43: 2457-2463; Porcari et al., 1999, NucleosidesNucleotides, 18: 2475-2497; Porcari et al., 2000, J Med Chem, 43:2438-2448; Porcari et al., 2003, Nucleosides Nucleotides Nucleic Acids,22: 2171-2193; Porcari et al., 2004, Nucleosides Nucleotides NucleicAcids 23: 31-39; Schweinsberg et al., 1981, Biochem Pharmacol 30:2521-2526; Smith et al., 2004, Bioorg Med Chem Lett 14: 3517-3520;Townsend et al., 1986, Nucleic Acids Symp Ser 1986: 41-44; and/or,Wotring et al., 1986, Cancer Treat Rep 70: 491-7.

5.3. Pharmaceutically Acceptable Salts and Prodrugs

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Pharmaceuticallyacceptable salts include those derived from pharmaceutically acceptableinorganic; or organic bases and acids. Suitable salts include thosederived from alkali metals such as potassium and sodium, alkaline earthmetals such as calcium and magnesium, among numerous other acids wellknown in the pharmaceutical art. In particular, examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids, which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including, sulfate,nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

Any of the nucleotides described herein can be administered as anucleotide prodrug to increase the activity, bioavailability, stabilityor otherwise alter the properties of the nucleoside. A number ofnucleotide prodrug ligands are known. In general, alkylation, acylationor other lipophilic modification of the mono, di or triphosphate of thenucleoside will increase the stability of the nucleotide. Examples ofsubstituent groups that can replace hydrogens on the phosphate moietyare alkyl, aryl, steroids, carbohydrates, including sugars,1,2-diacylglycerol and alcohols. Many are described in R. Jones and N.Bischofberger. Antiviral Research, 27 (1995) 1-17. Any of these can beused in combination with the disclosed nucleosides to achieve a desiredeffect.

In one embodiment, the triciribine or a related compound is provided as5′-hydroxyl lipophilic prodrug. Nonlimiting examples of U.S. patentsthat disclose suitable lipophilic substituents that can be covalentlyincorporated into the nucleoside, preferably at the 5′-OH position ofthe nucleoside or lipophilic preparations, include U.S. Pat. Nos.5,149,794 (Sep. 22, 1992, Yatvin, et al.); 5,194,654 (Mar. 16, 1993,Hostetler, et al.); 5,223,263 (Jun. 29, 1993, Hostetler, et al.);5,256,641 (Oct. 26, 1993, Yatvin, et al.); 5,411,947 (May 2, 1995,Hostetler, et al.); 5,463,092 (Oct. 31, 1995, Hostetler, et al.);5,543,389 (Aug. 6, 1996, Yatvin, et al.); 5,543,390 (Aug. 6, 1996,Yatvin, et al.); 5,543,391 (Aug. 6, 1996, Yatvin, et al.); and 5,554,728(Sep. 10, 1996, Basava, et al.), all of which are incorporated herein byreference.

Foreign patent applications that disclose lipophilic substituents thatcan be attached to the triciribine or a related compound s of thepresent invention, or lipophilic preparations, include WO 89/02733, WO90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO/15132,EP 0 350 287, EP 93917054.4, and WO 91/19721.

Additional nonlimiting examples of derivatives of triciribine or arelated compound s are those that contain substituents as described inthe following publications. These derivatized triciribine or a relatedcompound s can be used for the indications described in the text orotherwise as antiviral agents, including as anti-HIV or anti-HBV agents.Ho, 1973, Cancer Res 33: 2816-2820; Holy, 1993, Isopolarphosphorous-modified nucleotide analogues, in: De Clercq (ed.), Advancesin Antiviral Drug Design, Vol. I, JAI Press, pp. 179-231; Hong et al.,1976, Biochem Biophys Rs Commun 88: 1223-1229; Hong et al, 1980, J MedChem 28: 171-177; Hostetler et al., 1990, J Biol Chem 266: 11714-11717;Hostetler et al., 1994, Antiviral Res 24: 59-67; Hostetler et al., 1994,Antimicrobial Agents Chemother 38: 2792-2797; Hunston et al., 1984, JMed Chem 27: 440-444; Ji et al., 1990, J Med Chem 33: 2264-2270; Joneset al., 1984, J Chem Soc Perkin Trans I: 1471-1474; Juodka et al., 1974,Coll Czech Chem Comm 39: 363-968; Kataoka et al., 1989, Nucleic AcidsRes Sym Ser 21: 1-2; Kataoka et al., 1991, Heterocycles 32: 1351-1356;Kinchington et al., 1992, Antiviral Chem Chemother 3: 107-112; Kodama etal., 1989, Jpn J Cancer Res 80: 679-685; Korty et al., 1979,Naunyn-Schmiedeberg's Arch Pharmacol 310: 103-111; Kumar et al., 1990, JMed Chem 33: 2368-2375; LeBec et al., 1991, Tetrahedron Lett 32:6553-6556; Lichienstein et al., 1960, J Biol Chem 235: 457-465; Luethyet al., 1981, Mitt Geg Lebensmittelunters Hyg 72: 131-133 (Chem. Abstr.95, 127093); McGuigan et al., 1989, Nucleic Acids Res 17: 6065-6075;McGuigan et al., 1990, Antiviral Chem Chemother 1: 107-113; McGuigan etal., 1990, Antiviral Chem Chemother 1: 355-360; McGuigan et al., 1990,Antiviral Chem Chemother 1: 25-33; McGuigan et al., 1991, Antiviral Res15: 255-263; McGuigan et al., 1992, Antiviral Res 17: 311-321; McGuiganet al., 1993, Antiviral Chem Chemother 4: 97-101; McGuigan et al., 1993,J Med Chem 36: 1048-1052.

Alkyl hydrogen phosphonate derivatives of the anti-HIV agent AZT may beless toxic than the parent nucleoside analogue. Antiviral Chem Chemother5: 271-277; Meyer et al., 1973, Tetrahedron Lett 269-272; Nagyvary etal., 1973, Biochem Biophys Res Commun 55: 1072-1077; Namane et al.,1992, J Med Chem 35: 3939-3044; Nargeot et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80: 2395-2399; Nelson et al., 1987, J Am Chem Soc 109:4058-4064; Nerbonne et al., 1984, Nature 301: 74-76; Neumann et al.,1989, J Am Chem Soc 111: 4270-4277; Ohno et al., 1991, Oncology 48:451-455. Palomino et al., 1989, J Med Chem 32: 622-625; Perkins et al.,1993, Antiviral Res 20(Supp. I): 84; Piantadosi et al., 1991, J Med Chem34: 1408-1414; Pompon et al., 1994, Antiviral Chem Chemother 5: 91-98;Postemark, 1974, Anu Rev Pharmacol 14: 23-33; Prisbe et al., 1986, J MedChem 29: 671-675; Pucch et al., 1993, Antiviral Res 22: 155-174; Pugaevaet al., 1969, Gig Trf Prof Zabol 13: 47-48 (Chem. Abstr. 72, 212);Robins, 1984, Pharm Res 11-18; Rosowsky et al., 1982, J Med Chem 25:171-178; Ross, 1961, Biochem Pharm 8: 235-240; Ryu et al., 1982, J MedChem 25: 1322-1329; Saffhill et al., 1986, Chem Biol Interact 57:347-355; Saneyoshi et al., 1980, Chem Pharm Bull 28: 2915-2923; Sastryet al., 1992, Mol Pharmacol 41: 441-445; Shaw et al., 1994, 9th AnnualAAPS Meeting, San Diego, Calif. (Abstract); Shuto et al., 1987,Tetrahedron Lett 28: 199-202; Shuto et al., 1988; Chem Pharm Bull 36:209-217. One preferred phosphate prodrug group is the S-acyl-2-thioethylgroup, also referred to as “SATE.”

Additional examples of prodrugs that can be used are those described inthe following patents and patent applications: U.S. Pat. Nos. 5,614,548,5,512,671, 5,770,584, 5,962,437, 5,223,263, 5,817,638, 6,252,060,6,448,392, 5,411,947, 5,744,592, 5,484,809, 5,827,831, 5,696,277,6,022,029, 5,780,617, 5,194,654, 5,463,092, 5,744,461, 4,444,766,4,562,179, 4,599,205, 4,493,832, 4,221,732, 5,116,992, 6,429,227,5,149,794, 5,703,063, 5,888,990, 4,810,697, 5,512,671, 6,030,960,2004/0259845, 6,670,341, 2004/0161398, 2002/082242, 5,512,671,2002/0082242, and or PCT Publication Nos WO 90/11079, WO 96/39197,and/or WO 93/08807.

5.4. In Vivo Efficacy/Dosing Regimens

In another aspect of the present invention, dosing regimens are providedthat limit the toxic side effects of TCN and related compounds. In oneembodiment, such dosing regimens minimize the following toxic sideeffects, including, but not limited to hepatoxicity, thrombocytopenia,hyperglycemia, vomiting, hypocalcemia, anemia, hypoalbunemia,myelosuppression, hypertriglyceridemia, hyperamylasemia, diarrhea,stomachitis and/or fever.

In another embodiment, the administration of TCN, TCN-P or relatedcompounds and bortezomib and derivatives thereof analogs provides atleast a partial or complete response in vivo in at least 15-20% of thesubjects. In particular embodiments, a partial response can be at least15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80 or 85% regression ofthe tumor. In other embodiments, this response can be evident in atleast 15, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85 or 90%of the subjects treated with the therapy. In further embodiments, suchresponse rates can be obtained by any therapeutic regimen disclosedherein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject an effectiveamount of TCN, TCN-P or a related compound and bortezomib andderivatives thereof analogs according to a dosing schedule that includesadministering the triciribine compound and/or the bortezomib or salt orderivatives thereof one time per week for three weeks followed by a oneweek period wherein the drug is not administered (i.e., via a 28 daycycle). In other embodiments, such 28 day cycles can be repeated atleast 2, 3, 4, or 5 times or until regression of the tumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the triciribine compoundand/or the bortezomib and derivatives thereof is not administered. Inother embodiments, such 42 day cycles can be repeated at least 2, 3, 4,or 5 times or until regression of the tumor is evident. In a particularembodiment, less than 50, less than 25 or less than 10 mg/m² of TCN,TCN-P. TCN-PM or a related compound can be administered according to a42 day cycle. In other particular embodiments, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 mg/m² of TCN, TCN-P, TCN-PM or a related compound can beadministered according to a 42 day cycle. In another particularembodiment, about 0.1 mg/m² to about 50 mg/m² of bortezomib or aderivative thereof is administered. In a particular embodiment, 0.1,0.5, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mg/m² of bortezomib or a saltthereof can be administered according to a 42 day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P, TCN-PM or a related compound and less than about 30 mg ofbortezomib and derivatives thereof analogs one time per week. Inparticular embodiments, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/m² of TCN, TCN-P, TCN-PM or arelated compound as disclosed herein can be administered one time perweek In another particular embodiment, 0.1, 0.5, 1, 5, 10, 15, 20, 25,30, 35, or 40 mg/m² of bortezomib or a derivative thereof can beadministered one time per week.

In embodiments of the present invention, the compounds disclosed hereincan be administered simultaneously as a single bolus dose over a shortperiod of time, for example, about 5, 10, 15, 20, 30 or 60 minutes. Infurther embodiments, dosing schedules are provided in which thecompounds are administered simultaneously via continuous infusion for atleast 24, 48, 72, 96, or 120 hours. In certain embodiments, theadministration of the triciribine compound and/or the bortezomib andderivatives thereof analogs via continuous or bolus injections can berepeated at a certain frequency at least: once a week, once every twoweeks, once every three weeks, once a month, once every five weeks, onceevery six weeks, once every eight weeks, once every ten weeks and/oronce every twelve weeks. The type and frequency of administrations canbe combined in any manner disclosed herein to create a dosing cycle. Thetriciribine compound and/or the bortezomib and derivatives thereofanalogs can be repeatedly administered via a certain dosing cycles, forexample as a bolus injection once every two weeks for three months. Thedosing cycles can be administered for at least: one, two three, fourfive, six, seven, eight, nine, ten, eleven, twelve, eighteen or twentyfour months. Alternatively, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,15 or 20 dosing cycles can be administered to a patient. The triciribinecompound and/or the bortezomib and derivatives thereof analogs can beadministered according to any combination disclosed herein, for example,the triciribine compound and/or the bortezomib and derivatives thereofanalogs can be administered once a week every three weeks for 3 cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which thetriciribine compound and/or the bortezomib and derivatives thereofanalogs are not administered.

The TCN, TCN-P, TCN-PM and related compounds and bortezomib andderivatives thereof analogs as disclosed herein can be administered topatients in an amount that is effective in causing tumor regression. Theadministration of TCN, TCN-P, TCN-PM or related compounds and bortezomiband derivatives thereof analogs can provide at least a partial, such asat least 15, 20 or 30%, or complete response in vivo in at least 15-20%of the subjects. In certain embodiments, at least 2, 5, 10, 15, 20, 30or 50 mg/m² of a triciribine compound disclosed herein can beadministered to a subject. In certain embodiments, at least about 0.5,1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 150, 165, 175, 200, 250, 300, or 350 mg/m² of TCN, TCN-P,TCN-PM or a related compound disclosed herein can be administered to asubject. In certain embodiments, 1, 5, 10, 15, 20, 25, 30, 35, or 40mg/m² of bortezomib can be administered to a subject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than about 20 mg/m² ofTCN and related compounds and less than about 3.0 mg of bortezomibeither concurrently, sequentially, or conducted over a period of time.In one embodiment, less than 20 mg/m² of TCN or related compounds can beadministered once a week concurrently with less than about 10 mg/m² ofbortezomib. In another embodiment, less than 20 mg/m² of TCN or relatedcompounds can be administered once a week and less than about 30 mg ofbortezomib can be administered the following week.

In further embodiments, 2 mg/m², 5 mg/m², 10 mg/m² and/or 15 mg/m² ofTCN or a related compound and less than about 30, 25, 20, 15, 10, 5, 1,0.5, or 0.1 mg/m² of bortezomib or a salt or a derivative thereof can beadministered to a subject. In another embodiment, less than 10 mg/m² ofa triciribine compound and less than about 30 mg/m² of bortezomib can beadministered to a subject via continuous infusion for at least fivedays. The present invention provides for any combination of dosing type,frequency, number of cycles and dosage amount disclosed herein.

5.5. Screening of Patient Populations

In another aspect of the present invention, methods are provided toidentify cancers or tumors that are susceptible to the toxic effects oftriciribine (TCN) and related compounds. In one embodiment, methods areprovided to treat a cancer or tumor in a mammal by (i) obtaining abiological sample from the tumor; (ii) determining whether the cancer ortumor overexpresses Akt kinase or hyperactivated and phosphorylated Aktkinase, and (iii) treating live cancer or tumor with triciribine or arelated compound as described herein. In one embodiment, the biologicalsample can be a biopsy. In other embodiments, the biological sample canbe fluid, cells and/or aspirates obtained from the tumor or cancer.

The biological sample can be obtained according to any technique knownto one skilled in the art. In one embodiment, biopsy can be conducted toobtain the biological sample. A biopsy is a procedure performed toremove tissue or cells from the body for examination. Some biopsies canbe performed in a physician's office, while others need to be done in ahospital setting. In addition, some biopsies require use of ananesthetic to numb the area, while others do not require any sedation.In certain embodiments, an endoscopic biopsy can be performed. This typeof biopsy is performed through a fiberoptic endoscope (a long, thin tubethat has a close-focusing telescope on the end for viewing) through anatural body orifice (i.e., rectum) or a small incision (i.e.,arthroscopy). The endoscope is used to view the organ in question forabnormal or suspicious areas, in order to obtain a small amount oftissue for study. Endoscopic procedures are named for the organ or bodyarea to be visualized and/or treated. The physician can insert theendoscope into the gastrointestinal tract (alimentary tract endoscopy),bladder (cystoscopy), abdominal cavity (laparoscopy), joint cavity(arthroscopy), mid-portion of the chest (mediastinoscopy), or tracheaand bronchial system (laryngoscopy and bronchoscopy).

In another embodiment, a bone marrow biopsy can be performed. This typeof biopsy can be performed either from the sternum (breastbone) or theiliac crest hipbone (the bone area on either side of the pelvis on thelower back area). The skin is cleansed and a local anesthetic is givento numb the area. A long, rigid needle is inserted into the marrow, andcells are aspirated for study; this step is occasionally uncomfortable.A core biopsy (removing a small bone ‘chip’ from the marrow) may followthe aspiration.

In a further embodiment, an excisional or incisional biopsy can beperformed on the mammal. This type of biopsy is often used when a wideror deeper portion of the skin is needed. Using a scalpel (surgicalknife), a full thickness of skin is removed for further examination, andthe wound is sutured (sewed shut with surgical thread). When the entiretumor is removed, it is referred to as an excisional biopsy technique.If only a portion of the tumor is removed, it is referred to as anincisional biopsy technique. Excisional biopsy is often the methodusually preferred, for example, when melanoma (a type of skin cancer) issuspected.

In still further embodiments, a fine needle aspiration (FNA) biopsy canbe used. This type of biopsy involves using a thin needle to remove verysmall pieces from a tumor. Local anesthetic is sometimes used to numbthe area, but the test rarely causes much discomfort and leaves no scar.FNA is not, for example, used for diagnosis of a suspicious mole, butmay be used, for example, to biopsy large lymph nodes near a melanoma tosee if the melanoma has metastasized (spread). A computed tomographyscan (CT or CAT scan) can be used to guide a needle into a tumor in aninternal organ such as the lung or liver.

In other embodiments, punch shave and/or skin biopsies can be conducted.Punch biopsies involve taking a deeper sample of skin with a biopsyinstrument that removes a short cylinder, or “apple core,” of tissue.After a local anesthetic is administered, the instrument is rotated onthe surface of the skin until it cuts through all the layers, includingthe dermis, epidermis, and the most superficial parts of the subcutis(fat). A shave biopsy involves removing the top layers of skin byshaving it off. Shave biopsies are also performed with a localanesthetic. Skin biopsies involve removing a sample of skin forexamination under the microscope to determine if, for example, melanomais present. The biopsy is performed under local anesthesia.

In particular embodiment, methods are provided to determine whether thetumor overexpresses an Akt kinase. Akt kinase overexpression can referto the phosphorylation state of the kinase. Hyperphosphorylation of Aktcan be detected according to the methods described herein. In oneembodiment, a tumor biopsy can be compared to a control tissue. Thecontrol tissue can be a normal tissue from the mammal in which thebiopsy was obtained or a normal tissue from a healthy mammal. Akt kinaseoverexpression or hyperphosphorylation can be determined if the tumorbiopsy contains greater amounts of Akt kinase and/or Akt kinasephosphorylation than the control tissue, such as, for example, at leastapproximately 1.5, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5,4.75, 5, 5.5, 6, 7, 8, 9, or 10-fold greater amounts of Akt kinase thancontained in the control tissue.

In one embodiment, the present invention provides a method to detectaberrant Akt kinase expression in a subject or in a biological samplefrom the subject by contacting cells, cell extracts, serum or othersample from the subjects or said biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formation, wherein an elevated presence ofthe complex relative to a normal cell is indicative of an aberrant cellthat expresses or overexpresses Akt. In one example, cells or cellextracts can be screened immunologically for die presence of elevatedlevels of Akt kinase.

In an alternative embodiment, the aberrant expression of Akt in a cellis detected at the genetic level by screening for the level ofexpression of a gene encoding an Akt kinase wherein an elevated level ofa transcriptional expression product (i.e., mRNA) compared to a normalcell is indicative of an aberrant cell. In certain embodiments,real-time PCR as well as other PCR procedures can be used to determinetranscriptional activity. In one embodiment, mRNA can be obtained fromcells of a subject or from a biological sample from a subject and cDNAoptionally generated. The mRNA or cDNA can then be contacted with agenetic probe capable of hybridizing to and/or amplifying all or part ofa nucleotide sequence encoding Akt kinase or its complementarynucleotide sequence and then the level of the mRNA or cDNA can bedetected wherein the presence of elevated levels of the mRNA or cDNAcompared to normal controls can be assessed.

Yet another embodiment of the present invention contemplates the use ofan antibody, monoclonal or polyclonal, to Akt kinase in a quantitativeor semi-quantitative diagnostic kit to determine relative levels of Aktkinase in suspected cancer cells from a patient, which can include allthe reagents necessary to perform the assay. In one embodiment, a kitutilizing reagents and materials necessary to perform an ELISA assay isprovided. Reagents can include, for example, washing buffer, antibodydilution buffer, blocking buffer, cell staining solution, developingsolution, stop solution, anti-phospho-protein specific antibodies,anti-Pan protein specific antibodies, secondary antibodies, anddistilled water. The kit can also include instructions for use and canoptionally be automated or semi-automated or in a form which iscompatible with automated machine or software. In one embodiment, aphosphor-ser-473 Akt antibody that detects the activated form of AKT(Akt phosphorylated at serine 474) can be utilized as the antibody in adiagnostic kit. See, for example, Yuan et al. (2000) “FrequentActivation of AKT2 and induction of apoptosis by inhibition ofphosphinositide-3-OH kinase/Akt pathway in human ovarian cancer,”Oncogene 19:2324-2330.

5.6. Akt Kinases

Akt, also named PKB³, represents a subfamily of the serine/threoninekinase. Three members, AKT1, AKT2, and AKT3, have been identified inthis subfamily. Akt is activated by extracellular stimuli in aPI3K-dependent manner (Datta, S. R., et al. Genes Dev. 13; 2905-2927,1999). Full activation of Akt requires phosphorylation of Thr^(30S) inthe activation loop and Ser⁴⁷³ in the C-terminal activation domain. Aktis negatively regulated by PTEN tumor suppressor. Mutations in PTEN havebeen identified in various tumors, which lead to activation of Aktpathway (Datta, S. R., et al. Genes Dev. 13: 2905-2927, 1999). Inaddition, amplification, overexpression and/or activation of Akt havebeen detected in a number of human malignancies (Datta, S. R., et al.Genes Dev. 13: 2905-2927, 1999, Cheng, J. Q., and Nicosia, S. V. AKTsignal transduction pathway in oncogenesis. In Schwab D, editor.Encyclopedic Reference of Cancer. Berlin Heidelberg and New York:Springer; 2001. pp 35-7). Ectopic expression of Akt, especiallyconstitutively active Akt, induces cell survival and malignanttransformation whereas inhibition of Akt activity stimulates apoptosisin a range of mammalian cells (Datta, S. R., eta). Genes Dev. 13:2905-2927, 1999, Cheng, J. Q., and Nicosia, S. V. AKT signaltransduction pathway in oncogenesis. In Schwab D, editor. EncyclopedicReference of Cancer. Berlin Heidelberg and New York: Springer; 2001. pp35-7, Sun, M., et al. Am. J. Path., 159: 431-437, 2001, Cheng, J. Q., etal. Oncogene, 14-2793-2801, 1997). Further, activation of Akt has beenshown to associate with tumor invasiveness and chemoresistance (West, K.A., et al. Drug Resist. Updat., 5: 234-248, 2002).

Activation of the Akt pathway plays a pivotal role in malignanttransformation and chemoresistance by inducing cell survival, growth,migration, and angiogenesis. The present invention provides methods todetermine levels of Akt kinase overexpression and/or hyperactivated andphosphorylated Akt kinase.

The Akt kinase can be any known Akt family kinase, or kinase relatedthereto, including, but not limited to Akt 1, Akt 2, Akt 3. The mRNA andamino acid sequences of human Akt1, Akt2, and Akt 3 are illustrated inFIGS. 6 a-c, 7 a-d, and 8 a-c, respectively.

5.7. Diagnostic Assays

Immunological Assays

In one embodiment, a method is provided for detecting the aberrantexpression of an Akt kinase in a cell in a mammal or in a biologicalsample from the mammal, by contacting cells, cell extracts or serum orother sample from the mammal or biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formations and determining whether anelevated presence of the complex relative to a normal cell is present.

The immunointeractive molecule can be a molecule having specificity andbinding affinity for an Akt kinase or its antigenic parts or itshomologs or derivatives thereof. In one embodiment, theimmunointeractive molecule can be an immunglobulin molecule. In otherembodiments, the immunointeractive molecules can be an antibodyfragments, single chain antibodies, and/or deimmunized moleculesincluding humanized antibodies and T-cell associated antigen-bindingmolecules (TABMs). In one particular embodiment, the antibody can be amonoclonal antibody. In another particular embodiment, the antibody canbe a polyclonal antibody. The immunointeractive molecule can exhibitspecificity for an Akt kinase or more particularly an antigenicdeterminant or epitope on an Akt kinase. An antigenic determinant orepitope on an Akt kinase includes that part of the molecule to which animmune response is directed. The antigenic determinant or epitope can bea B-cell epitope or where appropriate a T-cell epitope. In oneembodiment, the antibody is a phosphor-ser 473 Akt antibody.

One embodiment of the present invention provides a method for diagnosingthe presence of cancer or cancer-like growth in a mammal, in whichaberrant Akt activity is present, by contacting cells or cell extractsfrom die mammal or a biological sample from the subject with an Aktkinase-binding effective amount of an antibody having specificity forthe Akt kinase or an antigenic determinant or epitope thereon and thenquantitatively or qualitatively determining the level of an Aktkinase-antibody complex wherein the presence of elevated levels of saidcomplex compared to a normal cell is determined.

Antibodies can be prepared by any of a number of means known to oneskilled in the art. For example, for the detection of human Akt kinase,antibodies can be generally but not necessarily derived from non-humananimals such as primates, livestock animals (e.g. sheep, cows, pigs,goats, horses), laboratory test animals (e.g. mice, rats, guinea pigs,rabbits) and/or companion animals (e.g. dogs, cats). Antibodies may alsobe recombinantly produced in prokaryotic or eukaryotic host cells.Generally, antibody based assays can be conducted in vitro on cell ortissue biopsies. However, if an antibody is suitably deimmunized or, inthe case of human use, humanized, then the antibody can be labeled with,for example, a nuclear tag, administered to a patient and the site ofnuclear label accumulation determined by radiological techniques. TheAkt kinase antibody can be a cancer targeting agent. Accordingly,another embodiment of the present invention provides deimmunized formsof the antibodies for use in cancer imaging in human and non-humanpatients.

In general, for the generation of antibodies to an Akt kinase, theenzyme is required to be extracted from a biological sample whether thisbe from animal including human tissue or from cell culture if producedby recombinant means. The Akt kinase can be separated from thebiological sample by any suitable means. For example, the separation maytake advantage of any of the Akt kinase's surface charge properties,size, density, biological activity and its affinity for another entity(e.g. another protein or chemical compound to which it binds orotherwise associates). Thus, for example, separation of the Akt kinasefrom the biological fluid can be achieved by any ofultra-centrifugation, ion-exchange chromatography (e.g. anion exchangechromatography, cation exchange chromatography), electrophoresis (e.g.polyacrylamide gel electrophoresis, isoelectric focussing), sizeseparation (e.g., gel filtration, ultra-filtration) andaffinity-mediated separation (e.g. immunoaffinity separation including,but not limited to, magnetic bead separation such as Dynabead(trademark) separation, immunochromatography, immuno-precipitation). Theseparation of Akt kinase from the biological fluid can preserveconformational epitopes present on the kinase and, thus, suitably avoidstechniques that cause denaturation of the enzyme. In a furtherembodiment, the kinase can be separated from the biological fluid usingany of affinity separation, gel filtration and/or ultra-filtration.

Immunization and subsequent production of monoclonal antibodies can becarried out using standard protocols known in the art, such as, forexample, described by Kohler and Milstein (Kohler and Milstein, Nature256: 495-499, 1975; Kohler and Milstein, Eur. J. Immunol. 6(7): 511-519,1976), Coligan et al. (“Current Protocols in Immunology, John Wiley &Sons, Inc., 1991-1997) or Toyama et al. (Monoclonal Antibody, ExperimentManual”, published by Kodansha Scientific, 1987). Essentially, an animalis immunized with an Akt kinase-containing biological fluid or fractionthereof or a recombinant form of Akt kinase by standard methods toproduce antibody-producing cells, particularly antibody-producingsomatic cells (e.g. B lymphocytes). These cells can then be removed fromthe immunized animal for immortalization. In certain embodiment, afragment of an Akt kinase can be used to the generate antibodies. Thefragment can be associated with a carrier. The carrier can be anysubstance of typically high molecular weight to which a non- or poorlyimmunogenic substance (e.g. a hapten) is naturally or artificiallylinked to enhance its immunogenicity.

Immortalization of antibody-producing cells can be carried out usingmethods which are well-known in the art. For example, theimmortalization may be achieved by the transformation method usingEpstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121: 140,1986). In another embodiment, antibody-producing cells are immortalizedusing the cell fusion method (described in Coligan et al., 1991-1997,supra), which is widely employed for the production of monoclonalantibodies. In this method, somatic antibody-producing cells with thepotential to produce antibodies, particularly B cells, are fused With amyeloma cell line. These somatic cells may be derived from the lymphnodes, spleens and peripheral blood of primed animals, preferably rodentanimals such as mice and rats. In a particular embodiment, mice spleencells can be used. In other embodiments, rat, rabbit, sheep or goatcells can also be used. Specialized myeloma cell lines have beendeveloped from lymphocytic tumours for use in hybridoma-producing fusionprocedures (Kohler and Milstein 1976, supra; Shulman et al., Nature 276:269-270, 1978; Volk et al., J. Virol. 42(1): 220-227, 1982). Manymyeloma cell lines can also be used for the production of fused cellhybrids, including, e.g. P3.times.63-Ag8, P3.times.63-AG8.653,P3/NS1-Ag-4-1 (NS-1), Sp2/0-Ag14; and S194/5.XXO.Bu.1. TheP3.times.63-Ag8 and NS-1 cell lines have been described by Kohler andMilstein (1976, supra). Shulman et al. (1978, supra) developed theSp2/0-Ag14 myeloma line. The S194/5.XXO.Bu.1 line was reported byTrowbridge (J. Exp. Med. 148(1): 313-323, 1978). Methods for generatinghybrids of antibody-producing spleen or lymph node cells and myelomacells usually involve mixing somatic cells with myeloma cells in a 10:1proportion (although the proportion may vary from about 20:1 to about1:1), respectively, in the presence of an agent or agents (chemical,viral or electrical) that promotes the fusion of cell membranes. Fusionmethods have been described (Kohler and Milstein, 1975, supra; Kohlerand Milstein, 1976, supra; Gefter et al., Somatic Cell Genet. 3:231-236, 1977; Volk et al., 1982, supra). The fusion-promoting agentsused by those investigators were Sendai virus and polyethylene glycol(PEG). In certain embodiments, means to select the fused cell hybridsfrom the remaining unfused cells, particularly the unfused myelomacells, are provided. Generally, the selection of fused cell hybrids canbe accomplished by culturing the cells in media that support the growthof hybridomas but prevent the growth of the unfused myeloma cells, whichnormally would go on dividing indefinitely. The somatic cells used inthe fusion do not maintain long-term viability in in vitro culture andhence do not pose a problem. Several weeks are required to selectivelyculture the fused cell hybrids. Early in this time period, it isnecessary to identify those hybrids which produce the desired antibody,so that they may subsequently be cloned and propagated. Generally,around 10% of the hybrids obtained produce the desired antibody,although a range of from about 1 to about 30% is not uncommon. Thedetection of antibody-producing hybrids can be achieved by any one ofseveral standard assay methods, including enzyme-linked immunoassay andradioimmunoassay techniques as, for example, described in Kennet et al.(Monoclonal Antibodies and Hybridomas: A New Dimension in BiologicalAnalyses, pp. 376-384, Plenum Press, New York, 1980) and by FAGSanalysis (O'Reilly et al., Biotechniques 25: 824-830, 1998).

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A suspension of the hybridomacells can be injected into a histocompatible animal. The injected animalwill then develop tumours that secrete the specific monoclonal antibodyproduced by the fused cell hybrid. The body fluids of the animal, suchas serum or ascites fluid, can be tapped to provide monoclonalantibodies in high concentration. Alternatively, the individual celllines may be propagated in vitro in laboratory culture vessels. Theculture medium containing high concentrations of a single specificmonoclonal antibody can be harvested by decantation, filtration orcentrifugation, and subsequently purified.

The cell lines can then be tested for their specificity to detect theAkt kinase of interest by any suitable immunodetection means. Forexample, cell lines can be aliquoted into a number of wells andincubated and the supernatant from each well is analyzed byenzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodytechnique, or the like. The cell line(s) producing a monoclonal antibodycapable of recognizing the target LIM kinase but which does notrecognize non-target epitopes are identified and then directly culturedin vitro or injected into a histocompatible animal to form tumours andto produce, collect and purify the required antibodies.

The present invention provides, therefore, a method of detecting in asample an Akt kinase or fragment, variant or derivative thereofcomprising contacting the sample with an antibody or fragment orderivative thereof and detecting the level of a complex containing theantibody and Akt kinase or fragment, variant or derivative thereofcompared to normal controls wherein elevated levels of Akt kinase isdetermined. Any suitable technique for determining formation of thecomplex may be used. For example, an antibody according to theinvention, having a reporter molecule associated therewith, may beutilized in immunoassays. Such immunoassays include but are not limitedto radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs)immunochromatographic techniques (ICTs), and Western blotting which arewell known to those of skill in the art. Immunoassays can also includecompetitive assays. The present invention encompasses qualitative andquantitative immunoassays.

Suitable immunoassay techniques are described, for example, in U.S. Pat.Nos. 4,016,043; 4,424,279; and 4,018,653. These include both single-siteand two-site assays of the non-competitive types, as well as thetraditional competitive binding assays. These assays also include directbinding of a labeled antigen-binding molecule to a target antigen.

The invention further provides methods for quantifying Akt proteinexpression and activation levels in cells or tissue samples obtainedfrom an animal, such as a human cancer patient or an individualsuspected of having cancer. In one embodiment, the invention providesmethods for quantifying Akt protein expression or activation levelsusing an imaging system quantitatively. The imaging system can be usedto receive, enhance, and process images of cells or tissue samples, thathave been stained with AKT protein-specific stains, in order todetermine the amount or activation level of AKT protein expressed in thecells or tissue samples from such an animal. In embodiments of themethods of the invention, a calibration curve of AKT 1 and AKT2 proteinexpression can be generated for at least two cell lines expressingdiffering amounts of AKT protein. The calibration curve can then used toquantitatively determine the amount of AKT protein that is expressed ina cell or tissue sample. Analogous calibration curves can be made foractivated AKT proteins using reagents specific for the activationfeatures. It can also be used to determine changes in amounts andactivation state of AKT before and after clinical cancer treatment.

In one particular embodiment of the methods of the invention, AKTprotein expression in a cell or tissue sample can be quantified using anenzyme-linked immunoabsorbent assay (ELISA) to determine the amount ofAKT protein in a sample. Such methods are described, for example, inU.S. Patent Publication No. 2002/0015974.

In other embodiments enzyme immunoassays can be used to detect the Aktkinase. In such assays, an enzyme is conjugated to the second antibody,generally by means of glutaraldehyde or periodate. The substrates to beused with the specific enzymes are generally chosen for the productionof, upon hydrolysis by the corresponding enzyme, a detectable colourchange. It is also possible to employ fluorogenic substrates, whichyield a fluorescent product rather than the chromogenic substrates. Theenzyme-labeled antibody can be added to the first antibody-antigencomplex, allowed to bind, and then the excess reagent washed away. Asolution containing the appropriate substrate can then be added to thecomplex of antibody-antigen-antibody. The substrate can react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of antigenwhich was present in the sample. Alternately, fluorescent compounds,such as fluorescein, rhodamine and the lanthanide, europium (EU), can bechemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. The fluorescent-labeled antibody is allowed to bind to thefirst antibody-antigen complex. After washing off the unbound reagent,the remaining tertiary complex is then exposed to light of anappropriate wavelength. The fluorescence observed indicates the presenceof the antigen of interest. Immunofluorometric assays (IFMA) are wellestablished in the art and are particularly useful for the presentmethod. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules can also be employed.

In a particular embodiment, antibodies to Akt kinase can also be used inELISA-mediated detection of Akt kinase especially in serum or othercirculatory fluid. This can be accomplished by immobilizing anti-Aktkinase antibodies to a solid support and contacting these with abiological extract such as serum, blood, lymph or other bodily fluid,cell extract or cell biopsy. Labeled anti-Akt kinase antibodies can thenbe used to detect immobilized Akt kinase. This assay can be varied manynumber of ways and all variations are encompassed by the presentinvention and known to one skilled in the art. This approach can enablerapid detection and quantitation of Akt kinase levels using, forexample, a serum-based assay.

In one embodiment, an Akt Elisa assay kit may be used in the presentinvention. For example, a Cellular Activation of Signaling ELISA kit forAkt S473 from SuperArray Bioscience can be utilized in the presentinvention. In one embodiment, the antibody can be an anti-pan antibodythat recognizes Akt S473. Elisa assay kit containing an anti-Aktantibody and additional reagents, including, but not limited to, washingbuffer, antibody dilution buffer, blocking buffer, cell stainingsolution, developing solution, stop solution, secondary antibodies, anddistilled water.

Nucleotide Detection

In another embodiment, a method to detect Akt kinases is provided bydetecting the level of expression in a cell of a polynucleotide encodingan Akt kinase. Expression of the polynucleotide can be determined usingany suitable technique known to one skilled in the art. In oneembodiment, a labeled polynucleotide encoding an Akt kinase can beutilized as a probe in a Northern blot of an RNA extract obtained fromthe cell. In other embodiments, a nucleic acid extract from an animalcan be utilized in concert with oligonucleotide primers corresponding tosense and antisense sequences of a polynucleotide encoding the kinase,or flanking sequences thereof, in a nucleic acid amplification reactionsuch as RT PCR. A variety of automated solid-phase detection techniquesare also available to one skilled in the art, for example, as describedby Fodor et al. (Science 251: 767-777, 1991) and Kazal et al. (NatureMedicine 2: 753-759, 1996).

In other embodiments, methods are provided to detect akt kinase encodingRNA transcripts. The RNA can be isolated from a cellular samplesuspected of containing Akt kinase RNA, e.g. total RNA isolated fromhuman cancer tissue. RNA can be isolated by methods known in the art,e.g. using TRIZOL reagent (GIBCO-BRL/Life Technologies, Gaithersburg,Md.). Oligo-dT, or random-sequence oligonucleotides, as well assequence-specific oligonucleotides can be employed as a primer in areverse transcriptase reaction to prepare first-strand cDNAs from theisolated RNA. Resultant first-strand cDNAs can then amplified withsequence-specific oligonucleotides in PCR reactions to yield anamplified product.

Polymerase chain reaction or “PCR” refers to a procedure or technique inwhich amounts of a preselected fragment of nucleic acid, RNA and/or DNA,are amplified as described, for example, in U.S. Pat. No. 4,683,195.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers. These primerswill be identical or similar in sequence to opposite strands of thetemplate to be amplified. PCR can be used to amplify specific RNAsequences and cDNA transcribed from total cellular RNA. See generallyMullis et al. (Quant. Biol. 51: 263, 1987; Erlich, eds., PCR Technology,Stockton Press, NY, 1989). Thus, amplification of specific nucleic acidsequences by PCR relies upon oligonucleotides or “primers” havingconserved nucleotide sequences wherein the conserved sequences arededuced from alignments of related gene or protein sequences, e.g. asequence comparison of mammalian Akt kinase genes. For example, oneprimer is prepared which is predicted to anneal to the antisense strandand another primer prepared which is predicted to anneal to the sensestrand of a cDNA molecule which encodes a Akt kinase. To detect theamplified product, the reaction mixture is typically subjected toagarose gel electrophoresis or other convenient separation technique andthe relative presence of the Akt kinase specific amplified DNA detected.For example, Akt kinase amplified DNA may be detected using Southernhybridization with a specific oligonucleotide probe or comparing itselectrophoretic mobility with DNA standards of known molecular weight.Isolation, purification and characterization of the amplified Akt kinaseDNA can be accomplished by excising or eluting the fragment from the gel(for example, see references Lawn et al., Nucleic Acids Res. 2: 6103,1981; Goeddel et al., Nucleic cids Res. 8: 4057-1980), cloning theamplified product into a cloning site of a suitable vector, such as thepCRII vector (Invitrogen), sequencing the cloned insert and comparingthe DNA sequence to the known sequence of LIM kinase. The relativeamounts of LIM kinase mRNA and cDNA can then be determined.

In one embodiment, real-time PCR can be used to determinetranscriptional levels of Akt nucleotides. Determination oftranscriptional activity also includes a measure of potentialtranslational activity based on available mRNA transcripts. Real-timePCR as well as other PCR procedures use a number of chemistries fordetection of PCR product including the binding of DNA bindingfluorophores, the 5′ endonuclease, adjacent liner and hairpinoligoprobes and the self-fluorescing amplicons. These chemistries andreal-time PCR in general are discussed, for example, in Mackay et al.,Nucleic Acids Res 30(6): 1292-1305, 2002; Walker, J. Biochem. Mol.Toxicology 15(3): 121-127, 2001; Lewis et al., J. Pathol. 195: 66-71,2001.

In an alternate embodiment, the aberrant expression of Akt can beidentified by contacting a nucleotide sequences isolated from abiological sample with an oligonucleotide probe having a sequencecomplementary to an Akt sequences selected from the nucleotide sequencesof FIGS. 6 a-c, 7 a-d, or 8 a-c, or fragment thereof, and then detectingthe sequence by hybridizing the probe to the sequence, and comparing theresults to a normal sample. The hybridization of the probe to thebiological sample can be detected by labeling the probe using anydetectable agent. The probe can be labeled for example, with aradioisotope, or with biotin, fluorescent dye, electron-dense reagent,enzyme, hapten or protein for which antibodies are available. Thedetectable label can be assayed by any desired means, includingspectroscopic, photochemical, biochemical, immunochemical,radioisotopic, or chemical means. The probe can also be detected usingtechniques such as an oligomer restriction technique, a dot blot assay,a reverse dot blot assay, a line probe assay, and a 5′ nuclease assay.Alternatively, the probe can be detected using any of the generallyapplicable DNA array technologies, including macroarray, microarray andDNA microchip technologies. The oligonucleotide probe typically includesapproximately at least 14, 15, 16, 18, 20, 25 or 28 nucleotides thathybridize to the nucleotides selected from FIGS. 6 a-c, 7 a-d, and 8a-c, or a fragment thereof. It is generally not preferred to use a probethat is greater than approximately 25 or 28 nucleotides in length. Theoligonucleotide probe is designed to identify an Akt nucleotidesequence.

Kinase Assays

The activity of the Akt kinases can be measured using any suitablekinase assay known in the art. For example, and not by way oflimitation, the methods described in Hogg et al (Oncogene 1994 9:98-96),Mills et al (J. Biol. Chem. 1992 267:16000-006) and Tomizawa et al 2001(FEBS Lett. 2001 492: 221-7), Schmandt et al, (J. Immunol. 1994,152:96-105) can be used. Further serine, threonine and tyrosine kinaseassays are described in Ausubel et al. (Short Protocols in MolecularBiology, 1999, unit 17.6).

Akt kinase assays can generally use an Akt polypeptide, a labeled donorsubstrate, and a receptor substrate that is either specific ornon-specific for Akt. In such assays Akt transfers a labeled moiety fromthe donor substrate to the receptor substrate, and kinase activity ismeasured by the amount of labeled moiety transferred from the donorsubstrate to the receptor substrate. Akt polypeptide can be producedusing various expression systems, can be purified from cells, can be inthe form of a cleaved or uncleaved recombinant fusion protein and/or canhave non-Akt polypeptide sequences, for example a His tag or.beta.-galactosidase at its N- or C-terminus. Akt activity can beassayed in cancerous cells lines if the cancerous cell lines are used asa source of the Akt to be assayed. Suitable donor substrates for Aktassays include any molecule that is susceptible to dephosphorylation byAkt., such as, for example include .gamma.-labeled ATP and ATP analogs,wherein the label is ³³P, ³²P, ³⁵S or any other radioactive isotope or asuitable fluorescent marker. Suitable recipient substrates for Aktassays include any polypeptide or other molecule dial is susceptible tophosphorylation by Akt. Recipient substrates can be derived fromfragments of in vivo targets of Akt. Recipient substrates fragments canbe 8 to 50 amino acids in length, usually 10 to 30 amino acids andparticularly of about 10, 12, 15, 18, 20 and 25 amino acids in length.Further recipient substrates can be determined empirically using a setof different polypeptides or other molecules. Targets of Recipientsubstrates for TTK can be capable of being purified from othercomponents of the reaction once the reaction has been performed. Thispurification is usually done through a molecular interaction, where therecipient substrates is biotinylated and purified through itsinteraction with streptavidin, or a specific antibody is available thatcan specifically recognize the recipient substrates. The reaction can beperformed in a variety of conditions, such as on a solid support, in agel, in solution or in living cells. The choice of detection methodsdepends on type of label used for the donor molecule and may include,for example, measurement of incorporated radiation or fluorescence byautoradiography, scintillation, scanning or fluorography.

6. METHODS OF TREATMENT

The compounds and pharmaceutical compositions provided herein can beused in the treatment of a condition including tumors, cancer, and otherdisorders associated with abnormal cell proliferation. In oneembodiment, the compounds of the present invention can be used to treata carcinoma, sarcoma, lymphoma, leukemia, and/or myeloma. In otherembodiments of the present invention, the compounds disclosed herein canbe used to treat solid tumors.

The compounds of the present invention can be used for the treatment ofcancer, such as, but not limited to cancer of the following organs ortissues: breast, prostate, lung, bronchus, colon, urinary, bladder,non-Hodgkin lymphoma, melanoma, kidney, renal, pancreas, pharnx,thyroid, stomach, brain, multiple myeloma, esophagus, liver,intrahepatic bile duct, cervix, larynx, acute myeloid leukemia, chroniclymphatic leukemia, soft tissue, such as heart, Hodgkin lymphoma,testis, small intestine, chronic myeloid leukemia, acute lymphaticleukemia, anus, anal canal, anorectal, thyroid, vulva, gallbladder,pleura, eye, nose nasal cavity, middle ear, nasopharnx, ureter,peritoneum, omentum, mesentery, and gastrointestineal, high gradeglioma, glioblastoma, colon, rectal, pancreatic, gastric cancers,hepatocellular carcinoma; head and neck cancers, carcinomas; renal cellcarcinoma; adenocarcinoma; sarcomas; hemangioendothelioma; lymphomas;leukemias, mycosis fungoides. In additional embodiments, the compoundsof the present invention can be used to treat skin diseases including,but not limited to, the malignant diseases angiosarcoma,hemangioendothelioma, basal cell carcinoma, squamous cell carcinoma,malignant melanoma and Kaposi's sarcoma, and the non-malignant diseasesor conditions such as psoriasis, lymphangiogenesis, hemangioma ofchildhood, Sturge-Weber syndrome, verruca vulgaris, neurofibromatosis,tuberous sclerosis, pyogenic granulomas, recessive dystrophicepidermolysis bullosa, venous ulcers, acne, rosacea, eczema, molluscumcontagious, seborrheic keratosis, and actinic keratosis.

Compositions including the compounds of the invention can be used totreat these cancers and other cancers at any stage from the discovery ofthe cancer to advanced stages. In addition, compositions includingcompounds of the invention can be used in the treatment of the primarycancer and metastases thereof.

In other embodiments of the invention, the compounds described hereincan be used for the treatment of cancer, including, but not limited tothe cancers listed in Table 1 below.

TABLE I Types of Cancer

Acute Lymphoblastic Leukemia, Adult Acute Lyinphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Adult Acute Myeloid Leukemia,Childhood Adrenocortical Carcinoma Adrenocortical Carcinoma, ChildhoodAIDS-Related Cancers AIDS-Related Lymphoma Anal Cancer Astrocytoma,Childhood Cerebellar Astrocytoma, Childhood Cerebral

Basal Cell Carcinoma Bile Duct Cancer, Extrahepatic Bladder CancerBladder Cancer, Childhood Bone Cancer, Osteosarcoma/Malignant FibrousHistiocytoma Brain Stem Glioma, Childhood Brain Tumor, Adult BrainTumor, Brain Stern Glioma, Childhood Brain Tumor, CerebellarAstrocytoma, Childhood Brain Tumor, Cerebral Astroytoma/MalignantGlioma; Childhood Brain Tumor, Ependymoma, Childhood Brain Tumor,Medulloblastoma, Childhood Brain Tumor, Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Brain Tumor, Visual Pathway andHypothalamic Glioma, Childhood Brain Tumor, Childhood Breast CancerBreast Cancer, Childhood Breast Cancer, Male BronchialAdenomas/Carcinoids, Childhood Burkitt's Lymphoma

Carcinoid Tumor, Childhood Carcinoid Tumor, Gastroint estinal Carcinomaof Unknown Primary Central Nervous System Lymphoma, Primary CerebellarAstrocytorna, Childhood Cerebral Astrocytoma/Malignant Glioma, ChildhoodCervical Cancer Childhood Cancers Chronic Lymphocytic Leukemia ChronicMyelogenous Leukemia Chronic Myeloproliferative Disorders Colon CancerColorectal Cancer; Childhood Cutaneous T-Cell Lymphoma, see MycosisFungoides and Sezary Syndrome

Endometrial Cancer Ependymoma Childhood Esophageal Cancer EsophagealCancer, Childhood Ewing's Family of Tumors Extracranial Germ Cell Tumor,Childhood Extragonadal Germ Cell Tumor Extrahepatic Bile Duet Cancer EyeCancer, Intraocular Melanoma Eye Cancer, Retinoblastoma

Gallbladder Cancer Gastric (Stomach) Cancer Gastric (Stomach) Cancer,Childhood Gastrointestinal Carcinoid Tumor Germ Cell Tumor,Extracranial, Childhood Germ Cell Tumor, Extragonadal Germ Cell Tumor,Ovarian Gestational Trophoblastic Tumor Glioma, Adult Glioma, ChildhoodBrain Stem Glioma, Childhood Cerebral Astrocytonia Glioma, ChildhoodVisual Pathway and Hypothalamic Skin Cancer (Melanoma) Skin Carcinoma,Merkel Cell Small Cell Lung Cancer Small Intestine Cancer Soft TissueSarcoma, Adult Soft Tissue Sarcoma, Childhood Squamous Cell Carcinoma,see Skin Cancer (non-Melanoma) Squamous Neck Cancer with Occult Primary,Metastatic Stomach (Gastric) Cancer Stomach (Gastric) Cancer, ChildhoodSupratentorial Primitive Neuroectodermal Tumors, Childhood

Hairy Cell Leukemia Head and Neck Cancer Hepatocellular (Liver) Cancer,Adult (Primary) Hepatocellular (Liver) Cancer, Childhood (Primary)Hodgkin's Lymphoma, Adult. Hodgkin's Lymphoma, Childhood Hodgkin'sLymphoma During Pregnancy Hypopharyngeal Cancer Hypothalamic and VisualPathway Glioma, Childhood

Intraocular Melanoma Islet Cell Carcinoma (Endocrine Pancreas).

Kaposi's Sarcoma Kidney (Renal Cell) Cancer Kidney Cancer, Childhood

Laryngeal Cancer Laryngeal Cancer, Childhood Leukemia, AcuteLymphoblastic, Adult Leukemia, Acute Lymphoblastic, Childhood Leukemia,Acute Myeloid, Adult Leukemia, Acute Myeloid, Childhood Leukemia,Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, B Cell Lipand Oral Cavity Cancer Liver Cancer, Adult (Primary) Liver Cancer,Childhood (Primary) Lung Cancer, Non-Small Cell Lung Cancer, Small CellLymphoma, AIDS-Related Lymphoma, Burkitt's Lymphoma, Cutanecius T-Cell,see Mycosis Fungoides and Sezary Syndrome Lymphoma, Hodgkin's, AdultLymphoma, Hodgkin's, Childhood Lymphoma, Hodgkin's During PregnancyLymphoma; Non-Hodgkin's, Adult Lymphoma, Non-Hodgkin's, ChildhoodLymphoma, Non-Hodgkin's During Prepancy Lymphoma, Primary CentralNervous System

Macroglobulinemia, Waldenström's Malignant Fibrous. Histiocytoma ofBone/Osteosarcoma Medulloblastoina, Childhood Melanoma Melanoma,Intraocular (Eye) Merkel Cell Carcinoma Mesothelioma, Adult MalignantMesothelioma, Childhood Metastatic Squamous Neck Cancer with OccultPrimary Multiple Endocrine Neoplasia Syndrome, Childhood MultipleMyeloma/Plasm Cell Neoplasm Mycosis Fungoides Myelodysplastic SyndromesMyelodysplastic/Myeloproliferative Diseases Myelogenous Leukemia,Chronic Myeloid Leukemia; Adult Acute Myeloid Leukemia, Childhood AcuteMyeloma, Multiple Myeloproliferative Disorders, Chronic

Nasal Cavity and Paranasal Sinus Cancer Nasopharyngeal CancerNasopharyngeal Cancer, Childhood Neuroblastoma Non-Hodgkin's Lymphoma,Adult Non-Hodgkin's Lyniphoma, Childhood Non-Hodgkin's Lymphoma DuringPregnancy Non-Small Cell Lung Cancer

Oral Cancer, Childhood Oral Cavity Cancer, Lip and Oropharyngeal CancerOsteosarcoma/Malignant Fibrous Histiocytoma of Bone Ovarian Cancer,Childhood Ovarian Epithelial Cancer Ovarian Germ Cell Tumor Ovarian LowMalignant Potential Tumor

Pancreatic Cancer Pancreatic Cancer, Childhood Pancreatic Cancer, IsletCell Paranasal Sinus and Nasal Cavity Cancer Parathyroid CancerPenile-Cancer Pheochromocytoma Pineoblastoma and SupratentorialPrimitive Neuroectodermal Tumors, Childhood Pituitary Tumor Plasma CellNeoplasm/Multiple Myeloma Pleuropulmonary Blastoma Pregnancy and BreastCancer Pregnancy and Hodgkin's. Lymphoma Pregnancy and Non-Hodgkin'sLymphoma Primary Central Nervous System Lymphoma Prostate Cancer

Rectal Cancer Renal Cell (Kidney) Cancer Renal Cell (Kidney) Cancer,Childhood Renal Pelvis and Ureter, Transitional Cell CancerRetinoblastorna Rhabdomyosarcoma, Childhood

Salivary Gland Cancer Salivary Gland Cancer, Childhood Sarcoma, Ewing'sFamily of Tumors Sarcoma, Kaposi's Sarcoma, Soft Tissue, Adult Sarcoma,Soft Tissue, Childhood Sarcoma, Uterine Sezary Syndrome Skin Cancer(non-Melanoma) Skin Cancer, Childhood

T-Cell Lymphoma, Cutaneous, see Mycosis Fungoides and Sezary SyndromeTesticular Cancer Thymoma, Childhood Thymoma and Thymic CarcinomaThyroid Cancer Thyroid Cancer, Childhood Transitional Cell Cancer of theRenal Pelvis and Ureter Trophoblastic Tumor, Gestational

Unknown Primary Site, Carcinoma of Adult Unknown Primaty Site, Cancerof, Childhood Unusual Cancers of Childhood Ureter and Renal Pelvis,Transitional Cell Cancer Urethral Cancer Uterine Cancer, EndometrialUterine Sarcoma

Vaginal Cancer Visual Pathway and Hypothalamic Glioma, Childhood VulvarCancer

Waldenström's Macroglobulinemia Wilms' Tumor

In further embodiments of the present invention, the compounds disclosedherein can be used in the treatment of angiogenesis-related diseases.

Antiangiogenic small molecules include thalidomide, which acts in partby inhibiting NFkB, 2-methoxyestradiol, which influences microtubuleactivation and hypoxia inducing factor (HIF1a) activation,cyclo-oxygenase 2 (COX2) inhibitors, and low doses of conventionalchemotherapeutic agents, including cyclophosphamide, and vinca alkaloids(vincristine, vinblastine) (D'Amato et al., 1994, Proc. Natl. Acad. Sci.U.S.A. 91: 3964-3968; D'Amato et al, 1994, Proc. Natl. Acad. Sci. U.S.A.91: 4082-4085). In addition, certain tyrosine kinase inhibitorsindirectly decrease angiogenesis by decreasing production of VEGF andother proangiogenic factors by tumor and stromal cells. These drugsinclude Herceptin, imatinib (Glivec), and Iressa (Bergers et al., 2003,J Clin Invest 111: 1287-1295; Ciardiello et al., 2001, Clin Cancer Res7: 1459-1465; Plum et al., 2003, Clin Cancer Res 9: 4619-4626).

Recently, angiogenesis inhibitors have moved from animal models to humanpatients. Angiogenesis inhibitors represent a promising treatment for avariety of cancers. Recently, Avastin a high affinity antibody againstvascular endothelial growth factor (VEGF), has been shown to prolonglife as a single agent in advanced renal cell carcinoma and prolong lifein combination with chemotherapy in advanced colon cancer (Yang et al.,2003, New Eng J Med 349: 427-434: Kabbinavar et al., 2003, J Clin Oncol21: 60-65).

Angiogenesis-related diseases include, but are not limited to,inflammatory, autoimmune, and infectious diseases;angiogenesis-dependent cancer, including, for example, solid tumors,blood born tumors such as leukemias, and tumor metastases; benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis;eczema; ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis;Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. In addition, compositions of this invention canbe used to treat diseases such as, but not limited to, intestinaladhesions, atherosclerosis, scleroderma, warts, and hypertrophic scars(i.e., keloids). Compositions of this invention can also be used in thetreatment of diseases that have angiogenesis as a pathologic consequencesuch as cat scratch disease (Rochele minalia quintosa), ulcers(Helobacter pylori), tuberculosis, and leprosy.

6.1. Treatment of Drug Resistant Tumors or Cancers

The invention provides compounds that can be used to treat drugresistant cancer, including the embodiments of cancers and thetriciribine compound and/or the bortezomib and derivatives thereofanalogs disclosed herein.

Multidrug resistance (MDR) occurs in human cancers and can be asignificant obstacle to the success of chemotherapy. Multidrugresistance is a phenomenon whereby tumor cells in vitro that have beenexposed to one cytotoxic agent develop cross-resistance to a range ofstructurally and functionally unrelated compounds. In addition, MDR canoccur intrinsically in some cancers without previous exposure tochemotherapy agents. Thus, in one embodiment, the present inventionprovides methods for the treatments of a patient with a drug resistantcancer, for examples, multidrug resistant cancer, by administration ofTCN, TCN-P, TCN-PM or a related compound and bortezomib and derivativesthereof analogs as disclosed herein. In certain embodiments, TCN, TCN-P,TCN-PM and related compounds and bortezomib and derivatives thereofanalogs can be used to neat cancers that are resistant to taxol alone,rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa).

In one embodiment TCN, TCN-P, TCN-PM or a related compound andbortezomib and derivatives thereof analogs as disclosed, herein can beused for the treatment of drug resistant cancers of the colon, bone,kidney, adrenal, pancreas, liver and/or any other cancer known in theart or described herein.

6.2. Combination Therapy

In one embodiment, the triciribine compounds and bortezomib andderivatives thereof analogs of the invention can be administeredtogether with other cytotoxic agents. In another embodiment, thetriciribine compounds and bortezomib and derivatives thereof analogs andcompositions thereof, when used in the treatment of solid tumors, can beadministered the use of radiation.

In another embodiment of the present invention, the triciribinecompounds and bortezomib and derivatives thereof analogs andcompositions disclosed herein can be combined with at least oneadditional chemotherapeutic agent. The additional agents can beadministered in combination or alternation with the compounds disclosedherein. The drugs can form part of the same composition, or be providedas a separate composition for administration at the same time or adifferent time.

In one embodiment, the triciribine compounds and bortezomib andderivatives thereof analogs disclosed herein can be combined withantiangiogenic agents to enhance their effectiveness, or combined withother antiangiogenic agents and administered together with othercytotoxic agents. In another embodiment, the triciribine compounds andbortezomib and derivatives thereof analogs and compositions, when usedin the treatment of solid tumors, can be administered with the agentsselected from, but not limited to IL-12, retinoids, interferons,angiostatin, endostatin, thalidomide, thrombospondin-1,thrombospondin-2, captopryl, anti-neoplastic agents such as alphainterferon, COMP (cyclophosphamide, vincristine, methotrexate andprednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP(prednisone, methotrexate (w/leucovin rescue), doxorubicin,cyclophosphamide, etoposide/mechlorethamine, vincristine, prednisone andprocarbazine), vincristine, vinblastine, angioinhibins, TNP-470,pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101,CM-101, Techgalan, thalidomide, SP-PG and radiation. In furtherembodiments, the compounds and compositions disclosed herein can beadministered in combination or alternation with, for example, drugs withantimitotic effects, such as those which target cytoskeletal elements,including podophylotoxins or vinca alkaloids (vincristine, vinblastine);antimetabolite drugs (such as 5-fluorouracil, cytarabine, gemcitabine,purine analogues such as pentostatin, methotrexate); alkylating agentsor nitrogen mustards (such as nitrosoureas, cyclophosphamide orifosphamide); drugs which target DNA such as the antracycline drugsadriamycin, doxorubicin, pharmorubicin or epirubicin; drugs which targettopoisomerases such as etoposide; hormones and hormone agonists orantagonists such as estrogens, antiestrogens (tamoxifen and relatedcompounds) and androgens, flutamide, leuprorelin, goserelin, cyprotroneor octreotide; drugs which target signal transduction in tumour cellsincluding antibody derivatives such as herceptin; alkylating drugs suchas platinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed below in Table 2.

TABLE 2 Chemotherapeutic Agents 13-cis-Retinoic Acid2-Amino-6-Mercaptopurine 2-CdA 2-Chlorodeoxyadenosine 5-fluorouracil5-FU 6-TG 6-Thioguanine 6-Mercaptopurine 6-MP Accutane Actinomycin-DAdriamycin Adrucil Agrylin Ala-Cort Aldesleukin Alemtuzumab AlitretinoinAlkaban-AQ Alkeran All-transretinoic acid Alpha interferon AltretamineAmethopterin Amifostine Aminoglutethimide Anagrelide AnandronAnastrozole Arabinosylcytosine Ara-C Aranesp Aredia Arimidex AromasinArsenic trioxide Asparaginase ATRA Avastin BCG BCNU BevacizumabBexarotene Bicalutamide BiCNU Blenoxane Bleomycin Busulfan Busulfex C225Calcium Leucovorin Campath Camptosar Camptothecin-11 Capecitabine CaraeCarboplatin Carmustine Carmustine wafer Casodex CCNU CDDP CeeNUCerubidine cetuximab Chlorambucil Cisplatin Citrovorum Factor CladribineCortisone Cosmegen CPT-11 Cyclophosphamide Cytadren CytarabineCytarabine liposomal Cytosar-U Cytoxan Dacarbazine DactinomycinDarbepoetin alfa Daunomycin Daunorubicin Daunorubicin hydrochlorideDaunorubicin liposomal DaunoXome Decadron Delta-Cortef DeltasoneDenileukin diftitox DepoCyt Dexamethasone Dexamethasone acetatedexamethasone sodium phosphate Dexasone Dexrazoxane DHAD DIC DiodexDocetaxel Doxil Doxorubicin Doxorubicin liposomal Droxia DTIC DTIC-DomeDuralone Efudex Eligard Ellence Eloxatin Elspar Emcyt Epirubicin Epoetinalfa Erbitux Erwinia L-asparaginase Estramustine Ethyol EtopophosEtoposide Etoposide phosphate Eulexin Evista Exemestane FarestonFaslodex Femara Filgrastim Floxuridine Fludara Fludarabine FluoroplexFluorouracil Fluorouracil (cream) Fluoxymesterone Flutamide Folinic AcidFUDR Fulvestrant G-CSF Getitinib Gemcitabine Gemtuzumab ozogamicinGemzar Gleevec Lupron Lupron Depot Matulane Maxidex MechlorethamineMechlorethamine Hydrochlorine Medralone Medrol Megace MegestrolMegestrol Acetate Melphalan Mercaptopurine Mesna Mesnex MethotrexateMethotrexate Sodium Methylprednisolone Mylocel Letrozole Neosar NeulastaNeumega Neupogen Nilandron Nilutamide Nitrogen Mustard NovaldexNovantrone Octreotide Octreotide acetate Oncospar Oncovin Ontak OnxalOprevelkin Orapred Orasone Oxaliplatin Paclitaxel Pamidronate PanretinParaplatin Pediapred PEG Interferon Pegaspargase PegfilgrastimPEG-INTRON PEG-L-asparaginase Phenylalanine Mustard Platinol Platinol-AQPrednisolone Prednisone Prelone Procarbazine PROCRIT ProleukinProlifeprospan 20 with Carmustine implant Purinethol RaloxifeneRheurnatrex Rituxan Rituximab Roveron-A (interferon alfa-2a) RubexRubidomycin hydrochloride Sandostatin Sandostatin LAR SargramostimSolu-Cortef Solu-Medrol STI -571 Streptozocin Tamoxifen Targretin TaxolTaxotere Temodar Temozolomide Teniposide TESPA Thalidomide ThalomidTheraCys Thioguanine Thioguanine Tabloid Thiophosphoamide ThioplexThiotepa TICE Toposar Topotecan Toremifene Bortezomib Tretinoin TrexallTrisenox TSPA VCR Velban Velcade VePesid Vesanoid Viadur VinblastineVinblastine Sulfate Vincasar Pfs Vincristine Vinorelbine Vinorelbinetartrate VLB VP-16 Vumon Xeloda Zanosar Zevalin Zinecard ZoladexZoledronic acid Zometa Gliadel wafer Glivec GM-CSF Goserelingranulocyte - colony stimulating factor Granulocyte macrophage colonystimulating factor Halotestin Herceptin Hexadrol HexalenHexamethylmelamine HMM Hycamtin Hydrea Hydrocort Acetate HydrocortisoneHydrocortisone sodium phosphate Hydrocortisone sodium succinateHydrocortone phosphate Hydroxyurea Ibritumomab Ibritumomab TiuxetanIdamycin Idarubicin Ifex IFN-alpha Ifosfamide IL-2 IL-11 Imatinibmesylate Imidazole Carboxamide Interferon alfa Interferon Alfa-2b (PEGconjugate) Interleukin-2 lnterlcukin-11 Intron A (interferon alfa-2b)Leucovorin Leukeran Leukine Leuprolide Leurocristine Leustatin LiposomalAra-C Liquid Pred Lomustine L-PAM L-Sarcolysin Meticorten MitomycinMitomycin-C Mitoxantrone M-Prednisol MTC MTX Mustargen Mustine MutamycinMyleran Iressa Irinotecan Isotretinoin Kidrolase Lanacort L-asparaginaseLCR

In certain embodiments, interferons (IFNs) can be used in combinationswith the compounds of the present invention. Suitable intereferonsinclude: interferon alpha-2a, interferon alpha-2b, pegylated interferonalpha, including interferon alpha-2a and interferon alpha 2b, interferonbeta, interferon gamma, interferon tau, interferon omega, INFERGEN(interferon alphacon-1) by InterMune, OMNIFERON (natural interferon) byViragens, ALBUFERON by Human Genome Sciences, REBIF (interferon beta-1a)by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alphaby Amarillo Biosciences, and interferon gamma, interferon tau, and/orinterferon gamma-1β by InterMune.

In one embodiment TCN, TCN-P, TCN-PM or a related compound and one ormore platinum compounds as disclosed herein can be used in combinationor alternation with additional chemotherapeutic agents, such as thosedescribed herein or in Table 3, for the treatment of drug resistantcancer, for example multiple drug resistant cancer. Drug resistantcancers can include cancers of the colon, bone, kidney, adrenal,pancreas, liver and/or any other cancer known in the art or describedherein. In one embodiment, the additional chemotherapeutic agent can bea P-glycoprotein inhibitor. In certain non-limiting embodiments, theP-glycoprotein inhibitor can be selected from the following drugs:verapamil, cyclosporin (such as cyclosporin A), tamoxifen, calmodulinantagonists, dexverapamil, dexniguldipine, valspodar (PSC 833),biricodar (VX-710), tariquidar (XR9576), zosuquidar (LY335979),laniquidar (R101933), and/or ONT-093.

7. PHARMACEUTICAL COMPOSITIONS

The compositions including triciribine compounds and bortezomib andderivatives thereof analogs can optionally be administered with apharmaceutical carrier or excipient. Pharmaceutical carriers suitablefor, administration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. The triciribine compounds and incombination with bortezomib and derivatives thereof analogs may beformulated as the sole pharmaceutically active ingredient in thecomposition or may be combined with bortezomib and derivatives thereofanalogs.

Compositions including the triciribine compounds and bortezomib andderivatives thereof analogs may be suitable for oral, rectal, nasal,topical (including buccal and sublingual), vaginal, or parenteral(including subcutaneous, intramuscular, subcutaneous, intravenous,intradermal, intraocular, intratracheal, intracisternal,intraperitoneal, and epidural) administration. Preferably thecompositions are administered intravenously.

The compositions may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association compositions ofthe present invention and pharmaceutical carriers or excipients.

The triciribine compounds and bortezomib and derivatives thereof analogsand compositions thereof can be formulated into suitable pharmaceuticalpreparations such as solutions, suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations orelixirs, for oral administration or in sterile solutions or suspensionsfor parenteral administration, as well as transdermal patch preparationand dry powder inhalers. In one embodiment, the triciribine compoundsdescribed above are formulated into pharmaceutical compositions usingtechniques and procedures well known in the art (see, e.g., AnselIntroduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).

In the compositions, effective concentrations of compounds orpharmaceutically acceptable derivatives thereof may be mixed withsuitable pharmaceutical carriers. The compounds of the invention may bederivatized as the corresponding salts, esters, enol ethers or esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs prior to formulation. The concentrationsof the compounds in the compositions are effective for delivery of anamount, upon administration, that treats, prevents, or ameliorates ofthe symptoms of the target disease or disorder. In one embodiment, thecompositions are formulated for single dosage administration. Toformulate a composition, the weight fraction of compound is dissolved,suspended, dispersed or otherwise mixed in a selected carrier at aneffective concentration such that the treated condition is relieved,prevented, or symptoms are ameliorated.

Compositions suitable for oral administration may be presented asdiscrete units such as, but not limited to, tablets, caplets, pills ordragees capsules, or cachets, each containing a predetermined amount ofthe compositions; as a powder or granules; as a solution or a suspensionin an aqueous liquid or a non-aqueous liquid; or as an oil-in-waterliquid emulsion or a water-in-oil emulsion or as a bolus, etc.

Liquid pharmaceutically administrate compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing a triciribinecompound and optional pharmaceutical adjuvants in a carrier, such as,for example, water, saline, aqueous dextrose, glycerol, glycols,ethanol, and the like, to thereby form a solution or suspension. Ifdesired, the pharmaceutical composition to be administered may alsocontain minor amounts of nontoxic auxiliary substances such as wettingagents, emulsifying agents, solubilizing agents, pH buffering agents,preservatives, flavoring agents, and the like, for example, acetate,sodium citrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents. Methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company; Easton, Pa., 15thEdition, 1975.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having triciribine compounds and bortezomib andderivatives thereof analogs of the present invention in an inert basissuch as gelatin and glycerin, or sucrose and acacia; and mouthwashes,having of the compositions of the present invention administered in asuitable liquid earlier.

The tablets, pills, capsules, troches and the like can contain of thefollowing ingredients, or compounds of a similar nature: a binder; alubricant; a diluent; a glidant; a disintegrating agent; a coloringagent; a sweetening agent; a flavoring agent; a wetting agent; an emeticcoating; and a film coating. Examples of binders includemicrocrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone,crospovidones, sucrose and starch paste. Lubricants include talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Diluents include, for example, lactose, sucrose, starch, kaolin, salt,mannitol and dicalcium phosphate. Glidants include, but are not limitedto, colloidal silicon dioxide. Disintegrating agents includecrosscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, for example, any of theapproved certified water soluble FD and C dyes, mixtures thereof; andwater insoluble FD and C dyes suspended on alumina hydrate. Sweeteningagents include sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include natural flavors extracted from plants such asfruits and synthetic blends of compounds which produce a pleasantsensation, such as, but not limited to peppermint and methyl salicylate.Wetting agents include propylene glycol monostearate, sorbitanmonooleate, diethylene glycol monolaurate and polyoxyethylene lauralether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

Compositions suitable for topical administration to the skin may bepresented as ointments, creams, gels, and pastes having the compositionsadministered in a pharmaceutical acceptable carrier.

Compositions for rectal administration may be presented as a suppositorywith a suitable base including, for example, cocoa butter or asalicylate.

Compositions suitable for nasal administration, when the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken, (i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose). Whenthe carrier is a liquid (for example, a nasal spray or as nasal drops),of the compositions can be admixed in an aqueous or oily solution, andinhaled or sprayed into the nasal passage.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining of the compositions and appropriate carriers.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats, and solutes which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. The compositions may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules, andtablets of the kind previously described above.

Pharmaceutical organic or inorganic solid or liquid carrier mediasuitable for enteral or parenteral administration can be used tofabricate the compositions. Gelatin, lactose, starch, magnesiumstearate, talc, vegetable and animal fats and oils, gum, polyalkyleneglycol, water, or other known carriers may all be suitable as carriermedia.

Compositions including triciribine compounds and bortezomib andderivatives thereof analogs may be used in combination withpharmaceutically acceptable carrier mediums and/or excipients. As usedherein, “pharmaceutically acceptable earner medium” includes any and allcarriers, solvents, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants, adjuvants,vehicles, delivery systems, disintegrants, absorbents, preservatives,surfactants, colorants, flavorants, or sweeteners and the like, assuited to the particular dosage form desired.

Additionally, the compositions including triciribine compounds andbortezomib and derivatives thereof analogs may be combined withpharmaceutically acceptable excipients, and, optionally,sustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions. A “pharmaceutically acceptable excipient”includes a non-toxic solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

It will be understood, however, that the total daily usage of thecompositions will be decided by the attending physician within the scopeof sound medical judgment. The specific therapeutically effective doselevel for any particular host will depend upon a variety of factors,including for example, the disorder being treated and the severity ofthe disorder; activity of the specific composition employed; thespecific composition employed, the age, body weight, general health, sexand diet of the patient; the time of administration; route ofadministration; rate of excretion of the specific compound employed; theduration of the treatment; the triciribine compound and/or thebortezomib and derivatives thereof analogs used in combination orcoincidental with the specific composition employed; and like factorswell known in the medical arts. For example, it is well within the skillof the art to start doses of the composition at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved.

Compositions including triciribine compounds and bortezomib andderivatives thereof analogs are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. “Dosage unitform” as used herein refers to a physically discrete unit of thecomposition appropriate for the host to be treated. Each dosage shouldcontain the quantity of composition calculated to produce the desiredtherapeutic affect either as such, or in association with the selectedpharmaceutical carrier medium.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. For example, approximately 1-5 mg per day of acompound disclosed herein can reduce the volume of a solid tumor inmice.

The dosage will depend on host factors such as weight, age, surfacearea, metabolism, tissue distribution, absorption rate and excretionrate. In one embodiment, approximately 0.5 to 7 grams per day of atriciribine compound disclosed herein may be administered to humans.Optionally, approximately 1 to 4 grams per day of the compound can beadministered to humans. In certain embodiments 0.001-5 mg/day isadministered to a human. The therapeutically effective dose level willdepend on many factors as noted above. In addition, it is well withinthe skill of the art to start doses of the composition at relatively lowlevels, and increase the dosage until the desired effect is achieved.

Compositions including triciribine compounds and bortezomib andderivatives thereof analogs may be used with a sustained-release matrix,which can be made of materials, usually polymers, which are degradableby enzymatic or acid-based hydrolysis or by dissolution. Once insertedinto the body, the matrix is acted upon by enzymes and body fluids. Asustained-release matrix for example is chosen from biocompatiblematerials such as liposomes, polylactides (polylactic acid),polyglycolide (polymer of glycolic acid), polylactide co-glycolide(copolymers of lactic acid and glycolic acid), polyanhydrides,poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is amatrix of one of either polylactide, polyglycolide, or polylactideco-glycolide (co-polymers of lactic acid and glycolic acid).

The triciribine compounds and bortezomib and derivatives thereof analogsmay also be administered in the form of liposomes. As is known in theart, liposomes are generally derived from phospholipids or other lipidsubstances. Liposomes are formed by mono- or multi-lamellar hydratedliquid crystals that are dispersed in an aqueous medium. Any non-toxic,physiologically-acceptable and metabolizable lipid capable of formingliposomes can be used. The liposome can contain, in addition tocompositions of the present invention, stabilizers, preservatives,excipients, and the like. Examples of lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art.

The triciribine compounds and bortezomib and derivatives thereof analogsmay be formulated as aerosols for application, such as by inhalation.These formulations for administration to the respiratory tract can be inthe form of an aerosol or solution for a nebulizer, or as a microfinepowder for insufflation, alone or in combination with an inert carriersuch as lactose. In such a case, the particles of the formulation will,in one embodiment, have diameters of less than 50 microns, in oneembodiment less than 10 microns.

Compositions including the triciribine compounds and bortezomib andderivatives thereof analogs may be used in combination with othercompositions and/or procedures for the treatment of the conditionsdescribed above. For example, a tumor may be treated conventionally withsurgery, radiation, or chemotherapy combined with compositions of thepresent invention and then compositions of the present invention may besubsequently administered to the patient to extend the dormancy ofmicrometastases and to stabilize, inhibit, or reduce the growth of anyresidual primary tumor.

7.1. Additional Embodiments

The pharmaceutical compositions including triciribine compounds andbortezomib and derivatives thereof analogs can be formulated accordingto known methods for preparing pharmaceutically useful compositions.Formulations are described in a number of sources which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., MackPublishing Company, 19^(th) ed.) describes formulations which can beused in connection with the subject invention. Formulations suitable foradministration include, for example, aqueous sterile injectionsolutions, which may contain antioxidants, buffers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and nonaqueous sterile suspensions whichmay include suspending agents and thickening agents. The formulationsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the condition of the sterileliquid earner, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The methods of the present invention, for example, for inhibiting thegrowth of a cancerous cell, can be advantageously combined with at leastone additional therapeutic method, including but not limited tochemotherapy, radiation therapy, therapy that selectively inhibits Rasoncogenic signaling, or any other therapy known to those of skill in thean of the treatment and management of cancer, such as administration ofan anti-cancer agent.

Administration of API-2 (triciribine) as a salt may be carried out.Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids which form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, andalpha-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion, Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The triciribine compounds and bortezomib and derivatives thereof analogscan be formulated as pharmaceutical compositions and administered to asubject, such as a human or veterinary patient, in a variety of formsadapted to the chosen route of administration, i.e., orally orparenterally, by intravenous, intramuscular, topical or subcutaneousroutes.

Thus, the triciribine compounds and bortezomib and derivatives thereofanalogs of the present invention may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle (i.e.,carrier) such as an inert diluent or an assimilable edible carrier. Theymay be enclosed in hard or soft shell gelatin capsules, may becompressed into tablets, or may be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, thecompounds may be combined with excipients and used in die form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active agent. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the compounds of the invention, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, thecompounds of the invention may be incorporated into sustained-releasepreparations and devices.

The triciribine compounds and bortezomib and derivatives thereof analogsmay also be administered intravenously or intraperitoneally by infusionor injection. Solutions of the active agents or their salts can beprepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powdersincluding the active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform must be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium including, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired panicle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating compounds ofthe invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the triciribine compounds and bortezomib andderivatives thereof analogs may be applied in pure-form, i.e., when theyare liquids. However, it will generally be desirable to administer themto the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds of the invention can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid earners to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the compounds of the invention to the skinare disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman(U.S. Pat. No. 4,820,508).

Useful dosages of the pharmaceutical compositions of the presentinvention can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

In one non-limiting embodiment, the concentration of the active agent ina liquid composition, such as a lotion, can be from about 0.1-25 wt-%,or from about 0.5-10 wt.-%. In one embodiment, the concentration in asemi-solid or solid composition such as a gel or a powder can be about0.1-5 wt.-%, preferably about 0.5-2.5 wt.-%. In one embodiment, singledosages for injection, infusion or ingestion will generally vary between5-1500 mg, and may be administered, i.e., 1-3 times daily, to yieldlevels of about 0.1-50 mg/kg, for adults. A non-limiting dosage of thepresent invention can be between 7.5 to 45 mg per clay, administeredorally, with appropriate adjustment for the body weight of anindividual.

Accordingly, the present invention includes a pharmaceutical compositionincluding triciribine compounds and bortezomib and derivatives thereofanalogs or pharmaceutically acceptable salts thereof, in combinationwith a pharmaceutically acceptable carrier. Pharmaceutical compositionsadapted for oral, topical or parenteral administration, including anamount of triciribine compounds and bortezomib and derivatives thereofanalogs or a pharmaceutically acceptable salt thereof, constitute apreferred embodiment of the invention. The dose administered to asubject, particularly a human, in the context of the present inventionshould be sufficient to affect a therapeutic response in the patientover a reasonable time frame. One skilled in the art will recognize thatdosage will depend upon a variety of factors including the condition ofthe animal, the body weight of the animal, as well as the severity andstage of the cancer.

A suitable dose is that which will result in a concentration of thetriciribine compounds and bortezomib and derivatives thereof analogs intumor tissue which is known to affect the desired response. Thepreferred dosage is die amount which results in maximum inhibition ofcancer cell growth, without unmanageable side effects. Administration ofAPI-2 (or a pharmaceutically acceptable salt thereof) can be continuousor at distinct intervals, as can be determined by a person of ordinaryskill in the art.

Mammalian species which benefit from the disclosed methods for theinhibition of cancer cell growth, include, but are not limited toprimates, such as apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. Theterms “patient” and “subject” are used herein interchangeably and areintended to include such human and non-human mammalian species.Likewise, in vitro methods of the present invention can be earned out oncells of such mammalian species.

Patients in need of treatment using the methods of the present inventioncan be identified using standard techniques known to those in themedical profession.

The following examples are offered by way of illustration and not by wayof limitation.

8. EXAMPLES 8.1. Example 1 In Vitro Screening

Cell Lines and NCI Diversity Set.

All cell lines can be purchased from ATCC or described previously (Chenget al., 1997, Oncogene 14: 2793-2801; West et al., 2002, Drug ResistUpdat 5: 234-248; Satyamoorthy et al., 2001, Cancer Res 61: 7318-7324).The NCI Structural Diversity Set is a library of 1,992 compoundsselected from the approximately 140,000-compound NCI drug depository.In-depth data on the selection, structures, and activities of thesediversity set compounds can be found on the NCI DevelopmentalTherapeutics Program web site.

Screening for Inhibition of Akt-transformed Cell Growth.

AKT2 transformed NIH3T3 cells or LXSN vector-transfected NIH3T3 controlcells (Cheng et al., 1997, Oncogene 14: 2793-2801) are plated into96-well tissue culture plate. Following treatment with 5 μM of NCIDiversity Set compound, cell growth can be detected with Cell Tier 96One Solution Cell Proliferation kit (Promega). Compounds that inhibitgrowth in AKT2-transformed but not LXSN-transfected NIH3T3 cells areconsidered as candidates of Akt inhibitor and subjected to furtheranalysis.

In vitro Protein Kinase, Cell Survival and Apoptosis Assays.

In vitro kinase is performed as previously described (see, for example,Jiang et al., 2000, Mol Cell Biol 20: 139-148). Cell survival is assayedwith MTS (Promega). Apoptosis was detected with annexin V, which isperformed as previously described (Jiang et al., 2000, Mol Cell Biol 20:139-148). Recombinant Akt and PDK1 are purchased from UpstateBiotechnology Inc.

Results

Identification of Small Molecule Inhibitor of Akt Signaling Pathway,API-2.

Frequent alterations of Akt has been detected in human cancer anddisruption of Akt pathway induces apoptosis and inhibits tumor growth(Jetzt et al., 2003, Cancer Res63: 697-706). Thus, Akt is considered asan attractive molecular target for development of novel cancertherapeutics. To identify small molecule inhibitor(s) of Akt, a chemicallibrary of 1,992-compounds from the NCI (the NCI Diversity Set) isevaluated for agents capable of inhibition of growth in AKT2-transformedbut not empty vector LXSN-transfected NIH3T3 cells. Repeated experimentsshowed that 32 compounds inhibited growth only in AKT2-transformaedcells. The most potent of these compounds, API-2 (NCI identifier: NSC154020), can suppress cell growth at a concentration of 50-nM. FIG. 1Ashows the chemical structure of API-2, which is also known astriciribine (Schweinsberg et al., 1981, Biochem Pharmacol 30:2521-2526). The fact that API-2 inhibits selectively AKT-2 transformedcells over untransformed parental cells prompted us to determine whetherAPI-2 is an inhibitor of AKT2 kinase. To this end, AKT2 isimmunoprecipitated with anti-AKT2 antibody from AKT-2 transformed NIH3T3cells following treatment with API-2. AKT2 immunoprecipitates wereimmunoblotted with anti-phospho-Akt antibodies. As shown in FIG. 1B,API-2 significantly inhibited AKT2 phosphorylation at both threonine-309and serine-474, which are required for full activation of AKT2 (Datta etal., 1999, Genes Dev 13: 2905-2927). As three isoforms of Akt share highhomology and similar structure, the effect of API-2 on their kinaseactivities is evaluated. HEK293 cells are transfected with HA-Akt1,-AKT2, and -AKT3, serum-starved overnight and treated with API-2 for 60min prior to EGF (50 ng/ml) stimulation. Triple experiments showed thatAPI-2 suppressed EGF-induced kinase activity and phosphorylation ofAkt1, AKT2 and AKT3 (FIG. 1C). However, kinase activity of recombinantconstitutively active AKT2 (Myr-AKT2) is not inhibited by API-2 in an invitro kinase reaction (FIG. 1D), suggesting that API-2 does not directlyinhibit Akt in vitro and that API-2 neither functions as ATP competitornor as the substrate competitor that binds to active site of Akt.

API-2 does not Inhibit Known Upstream Activators of Akt.

It has been well documented that Akt is activated by extracellularstimuli and intracellular signal molecules, such as active Ras and Src,through a PI3K-dependent manner. Therefore, API-2 inhibition of Aktcould result from targeting upstream molecule(s) of Akt. As PI3K andPDK1 are direct upstream regulators of Akt (Datta et al., 1999, GenesDev 13: 2905-2927), whether API-2 inhibits PI3K and/or PDK1 is examined.HEK293 cells are serum-starved and then can be treated with API-2 orPI3K inhibitor, wortmannin, for 30 min prior to EGF stimulation. PI3K isimmunoprecipitated with anti-p110α antibody. The immunoprecipitates aresubjected to in vitro PI3K kinase assay using PI-4-P as a substrate. Asshown in FIG. 2A, the EGF-induced PI3K activity is inhibited bywortmannin but not by API-2. To evaluate the effect of API-2 on PDK1, anassay in which recombinant PDK1 promotes the threonine-309phosphorylation of AKT2 peptides is used in the presence of lipidvesicles containing phosphotidylinositol. As shown in FIG. 2B, the assayis potently inhibited by the control PDK1 inhibitor staurosporine(IC50=5 nM). In contrast, API-2 displayed only 21% inhibition of theassay at the highest concentration tested (5.1 μM). To further evaluatethe effect of API-2 on PDK1 activation, the autophosphorylation level ofPDK1 at serine-241, a residue that is phosphorylated by itself and iscritical for its activity is examined (Datta et al., 1999, Genes Dev 13:2905-2927), following API-2 treatment of HEK293 cells. Triplicateexperiments show that phosphorylation levels of PDK1 are not inhibitedby API-2 (FIG. 2B). However, PI3K inhibitor wortmannin can inhibitEGF-stimulated PDK1 (FIG. 2B).

API-2 is Highly Selective for the Akt over PKC, PKA, SGK, STAT, JNK,p38, and ERK Signaling Pathways.

Akt belongs to AGC (PKA/PKG/PKC) kinase family, which also include PKA,PKC, serum- and glucocorticoid-inducible kinase (SGK), p90 ribosomal S6kinase, p70^(S6K), mitogen- and stress-activated protein kinase andPKC-related kinase. Among AGC kinase family, protein structures of PKA,PKC and SGK are more close to Akt kinase than other members. Therefore,next examined are the effects of API-2 on the enzymatic activities ofthese 3 kinases. HEK293 cells are transfected with HA-tagged PKA, PKCαor SGK. In vitro kinase assay and immunoblotting analysis show that thekinase activities of PKA and PKCα are inhibited by PKAI and Ro 31-8220,a PKC inhibitor, respectively, whereas API-2 exhibits no effect on theiractivities (FIGS. 2C and 2E). Further, serum-induced SGK kinase activityis attenuated by wortmannin but not by API-2 (FIG. 2D). In addition, itis determined whether API-2 has effect on other oncogenic survivalpathways. Western blotting analyses with commercially availableanti-phospho-antibodies reveals that phosphorylation levels of Stat3,JNK, p38 and Erk ½ were not affected by API-2 treatment (FIG. 2F). Thesedata indicate that API-2 specifically inhibits Akt signaling pathway.

API-2 Suppresses Cell Growth and Induces Apoptosis inAkt-Overexpressing/Activating Human Cancer Cell Lines.

The ability of API-2 to selectively inhibit the Akt pathway suggeststhat it should inhibit proliferation and/or induces apoptosispreferentially in those tumor cells with aberrant expression/activationof Akt. As activation of Akt in human malignancies commonly results fromoverexpression of Akt or PTEN mutations, API-2 is used to treat thecells that express constitutively active Akt, caused by overexpressionof AKT2 (OVCAR3, OVCAR8, PANC1 and AKT2-transformed NIH3T3) or mutationsof the PTEN gene (PC-3, LNCaP, MDA-MB-468), and cells that do not(OVCAR5, DU-145, T47D, COLO357 and LXSN-NIH3T3) as well as melanomacells that are activated by IGF-1 to activate Akt or do not respond togrowth stimulation by IGF-1 (Satyamoorthy et al., 2001, Cancer Res 61:7318-7324). Immunoblotting analysis showed that phosphorylation levelsof Akt are inhibited by API-2 only in the cells expressing elevated Aktor responding to IGF-1 simulation (FIG. 3A). Accordingly, API-2inhibited cell growth to a much higher degree inAkt-overexpressing/activating cells as compared to those with low levelsof Akt. As shown in FIG. 3B, API-2 treatment inhibited cellproliferation by approximate 50-60% in Akt-overexpressing/activatingcell lines, LNCaP, PC-3, OVCAR3, OVCA8, PANC1, MDA-MB-468, and WM35,whereas only by about 10-20% in DUI45, OVCAR5, COLO357, T47D and WM852cells, which exhibit low levels of Akt or do not respond to growthstimulation by IGF-1. Moreover, API-2 induces apoptosis by 8-fold(OVCAR3), 6-fold (OVCAR8), 6-fold (PANC1), and 3-fold (AKT2-NIH3T3). Nosignificant difference of apoptosis is observed between API-2 andvehicle (DMSO) treatment in OVCAR5, COLO357 and LXSN-NIH3T3 cells. Thus,API-2 inhibits cell growth and induces apoptosis preferentially in cellsthat express aberrant Akt.

API-2 Inhibits Downstream Targets of Akt.

It has been shown that Akt exerts its cellular effects throughphosphorylation of a number of proteins (Datta et al, 1999, Genes Dev13: 2905-2927). More than 20 proteins have been identified as Aktsubstrates, including the members of Forkhead protein family (FKHR, AFXand FKHRL1), tuberlin/TSC2, p70^(S6K), GSK-3β, p21^(WAF1/Cip1),p27^(kip1), MDM2, Bad, ASK1 and IKKα etc. It is next examined whetherAPI-2 inhibits downstream targets of Akt As anti-phospho-tuberlin, -Bad,-AFX, and -GSK-3β antibodies are commercially available, therefore, theeffect of API-2 on their phosphorylation induced by Akt was determined.Following API-2 (1 μM) treatment, OVCAR3 cells were lysed andimmunoblotted with the individual anti-phospho-antibody. FIG. 4A showsthat API-2 considerably inhibited the phosphorylation levels of tuberlinleading to stabilization and upregulation of tuberin (Dan et al., 2002,J Biol Chem 277: 35364-35370). The phosphorylation levels of Bad,GSK-3β, and AFX are partially attenuated by API-2. These data suggestthat API-2 induces cell death and cell growth arrest by inhibitingphosphorylation of its downstream targets. API-2 inhibition of Aktdownstream targets at different degrees could be due to the fact thatphosphorylation sites of these targets are also regulated by otherkinase(s), for instance, Bad serine-136 is phosphorylated by PAK1 inaddition to Akt (Schurmann et al., 2000, Mol Cell Biol 20: 453-461).

8.2 Example 2 Antitumor Activity in the Nude Mouse Tumor Xenograft Model

Tumor cells can be harvested, suspended in PBS, and can be injected s.c.into the right and left flanks (2×10⁶ cells/flank) of 8-week-old femalenude mice as reported previously (Sun et al., 1999, Cancer Res 59:4919-4926). When tumors reach about 100-150 mm³, animals are randomizedand dosed i.p. with 0.2 ml vehicle of the triciribine compound and/orone or more platinum compounds daily. Control animals receive DMSO (20%)vehicle, whereas treated animals can be injected with API-2 (1mg/kg/day) in 20% DMSO.

API-2 Inhibits the Growth of Tumors in Nude Mice that Overexpress Akt.

Frequent overexpression/activation and/or amplification of AKT1 and AKT2in human ovarian and pancreatic cancer was shown (Cheng et al., AKTsignal transduction pathway in oncogenesis, in Schwab D(ed.)Encyclopedic Reference of Cancer, Springer, pp 35-7). Inhibition of Aktpathway by inhibitors of PI3K, HSP70, Src and farnesyltransferaseresulted in cell growth arrest and induction of apoptosis (Solit et al.,2003, Cancer Res 63: 2139-2144; Xu et al., 2003, Cancer Res 63:7777-7784). A recent study showed that the tumor growth of xenograftswith elevated Akt was also significantly inhibited by intratumoralinjection of adenovirus of dominant negative Akt (Jetzt et al., 2003,Cancer Res 63: 697-706). Because API-2 inhibits Akt signaling andinduces apoptosis and cell growth arrest only in cancer cells withelevated levels of Akt (FIG. 3), the growth of tumors with elevatedlevels of Akt should be more sensitive to API-2 than that of tumors withlow levels of Akt in nude mice. To this end, s.c. Akt-overexpressingcells (OVCAR3, OVCAR8 and PANC-1) are s.c. implanted into the rightflank, and those cell lines that express low levels of Akt (OVCAR5 andCOLO357) into the left flank of mice. When the tumors reach an averagesize of about 100-150 mm³, the animals are randomized and treated i.p.with either vehicle or API-2 (1 mg/kg/day). As illustrated in FIG. 4B,OVCAR-5 and COLO357 tumors treated with vehicle grew to about 800-1,000mm³ 49 days after tumor implantation. OVCAR3, OVCAR8 and PANC1 tumorstreated with vehicle control grew to about 700-900 mm³ 49 days aftertumor implantation. API-2 inhibited OVCAR3, OVCAR8 and PANC1 tumorgrowth by 90%, 88% and 80%, respectively. In contrast, API-2 has littleeffect on the growth of OVCAR5 and COLO357 cells in nude mice (FIGS.4B-4D and data not shown). At dose 1 mg/kg/day, API-2 had no effects onblood glucose level, body weighty activity and food intake of mice. Intreated tumor samples, Akt activity was inhibited by API-2 withoutchange of total Akt content (FIG. 4E). Taken together, these resultsindicate that API-2 selectively inhibits the growth of tumors withelevated levels of Akt.

8.3 Example 3 TCN Directly Inhibits Wild Type Akt Kinase Activity

API-2 (TCN) can directly inhibit wild type Akt kinase activity inducedby PDK1 in vitro (FIG. 1). This result supports that API-2 is a directAkt inhibitor and that the underlying mechanism may be API-2 binding toPH domain and/or threonine-308 of Akt. An in vitro kinase assay isperformed with recombinant of PDK1 and Akt in a kinase buffer containingphosphatidylinositol-3,4,5-P3 (PIP3). API-2 and histone H2B assubstrate. After incubation of 30 min, the reactions were separated bySDS-PAGE and exposed in a film.

8.4 Example 4 TCN is Effective in Cancer Resistant Cells

The effects of TCN (API-2) are tested in cisplatin, paclitaxel, andtamoxifen resistant A270CP, C-13, OVCAR433 and MCF7/TAM cells. API-2overcame cisplatin, paclitaxel, and tamoxifen resistance in these cells.

This invention has been described with reference to its preferredembodiments. Variations and modifications of die invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

1-26. (canceled)
 27. A diagnostic method for treating a subject having atumor or cancer, which tumor or cancer overexpresses AKT kinasecomprising: A. obtaining a biological sample from the subject; B.determining whether the sample overexpresses AKT kinase; C. if saidsample overexpresses AKT kinase,
 1. administering to said subject: i. atleast one compound of formula I selected from the group consisting ofthe following compounds:

wherein each R₂′, R₃′, and R₅′ is independently hydrogen; optionallysubstituted phosphate or phosphonate; mono-, di-, or triphosphate; acyl;lower acyl; alkyl; lower alkyl; amide; sulfonate ester; alkyl sulfonateester; arylalkyl sulfonate ester; sulfonyl; methanesulfonyl; benzylsulfonyl, wherein the phenyl group of said benzyl is optionallysubstituted with one or more halo, hydroxyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate; optionally substitutedarylsulfonyl; a lipid; phospholipid; an amino acid; a carbohydrate; apeptide; or cholesterol; or other pharmaceutically acceptable leavinggroup that, in vivo, provides a compound of said formula I wherein R₂′,R₃′ or R₅′ is independently H or mono-, di- or tri-phosphate; whereinR^(x) and R^(y) are independently hydrogen; optionally substitutedphosphate; acyl; lower acyl; amide; alkyl; lower alkyl; aromatic;polyoxyalkylene; polyethyleneglycol; optionally substitutedarylsulfonyl; a lipid; a phospholipid; an amino acid; a carbohydrate; apeptide; or cholesterol; or other pharmaceutically acceptable leavinggroup; and wherein R₁ and R₂ each are independently H, optionallysubstituted straight chained, branched or cyclic alkyl, lower alkyl,alkenyl, or alkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl orheteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonyl, alkylsulfonyl, arylsulfonyl, or aralkylsulfonyl; ii. acompound of the formula V:

wherein R₁, R₂, R₃, R₄ and R₅ each are independently H, optionallyhalogenated, substituted straight chained, branched or cyclic alkyl(including lower alkyl), alkoxyl, alkenyl, or alkynyl, aryl, CO-alkyl,CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl,CO-aryloxyalkyl, CO-substituted aryl, sulfonyl, alkylsulfonyl,arylsulfonyl, aralkylsulfonyl; and iii. a pharmaceutically acceptablecarrier.
 28. The composition of claim 27, wherein the compound offormula I is triciribine.
 29. The composition of claim 27, wherein thecompound of formula I is triciribine phosphate.
 30. The composition ofclaim 27, wherein the compound of formula I is triciribine phosphonate.31. The composition of claim 27, wherein the compound of formula I ispresent in a dose amount of at least 20 mg/m².
 32. The composition ofclaim 27, wherein the compound of formula I is present in an amount ofat least 10 mg/m².
 33. The composition of claim 27, suitable forparenteral administration.
 34. The composition of claim 27, wherein theparenteral administration is intravenous administration.
 35. Thecomposition of claim 27, suitable for oral administration.
 36. Thecomposition of claim 27, suitable for topical administration.
 37. Thecomposition of claim 27, wherein the compound of formula V is:

or a salt thereof.
 38. The composition of claim 27, wherein the compoundof formula V or a salt thereof is present in an amount from about 1 mgto about 1000 mg.
 39. The composition of claim 27, wherein the compoundof formula V or a salt thereof is present in an amount from about 100 mgto about 500 mg.
 40. The composition of claim 27, wherein the compoundof formula V or a salt thereof is present in an amount from about 200 mgto about 450 mg.
 41. The composition of claim 27, wherein the compoundof formula V or a salt thereof is present in an amount of about 400 mg.42. The composition of claim 27, wherein the administration of acompound of formula I and the compound of formula V is a singlecomposition.
 43. The composition of claim 27, wherein the administrationof a compound of formula I and the compound of formula V is concurrentlyadministered.
 44. The composition of claim 27, wherein theadministration of a compound of formula I is followed by theadministration of the compound of formula V.
 45. The composition ofclaim 27, wherein the administration of the compound of formula V isfollowed by the administration of a compound of the compound of formulaV I.